Characterization of recombinant Aspergillus fumigatus mannitol-1-phosphate 5-dehydrogenase and its application for the stereoselective synthesis of protio and deuterio forms of d-mannitol 1-phosphate

Article abstract of DOI:10.1016/j.carres.2008.04.011

A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a ≈40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 °C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[²H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[²H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD⁺, producing (4S)-[²H]-NADH. Comparison of maximum initial rates for NAD⁺-dependent oxidation of protio and deuterio forms of d-mannitol 1-phosphate at pH 7.1 and 25 °C revealed a primary kinetic isotope effect of 2.9 ± 0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions.

Full text of DOI:10.1016/j.carres.2008.04.011

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1414–1423
Characterization of recombinant Aspergillus fumigatus
mannitol-1-phosphate 5-dehydrogenase and its application
for the stereoselective synthesis of protio and deuterio
forms of D-mannitol 1-phosphate
Stefan Krahulec,a Guilliano C. Armao,a Hansjo¨rg Weber,b Mario Klimaceka,*
and Bernd Nidetzkya,*
aInstitute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
bInstitute of Organic Chemistry, Graz University of Technology, Stremayrgasse 16, A-8010 Graz, Austria
Received 1 February 2008; received in revised form 2 April 2008; accepted 4 April 2008
Available online 10 April 2008
Abstract—A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpres-
sed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a ꢀ40-kDa mono-
mer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of D-mannitol 1-
phosphate and D-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 °C. NADP(H) showed a mar-
ginal activity. Hydrogen transfer from formate to D-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme
system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of D-mannitol 1-
phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation
of D-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%,
based on 200 mM of D-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of D-5-
[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing
(4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol
1-phosphate at pH 7.1 and 25 °C revealed a primary kinetic isotope effect of 2.9 0.2, suggesting that the hydride transfer was
strongly rate-determining for the overall enzymatic reaction under these conditions.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: D-Mannitol 1-phosphate; D-5-[2H]-Mannitol 1-phosphate; Mannitol-1-phosphate 5-dehydrogenase; Aspergillus fumigatus; Biocatalysis;
Stereoselective synthesis
1. Introduction
eukaryotes.1 Aspergillus niger utilizes mannitol to pro-
tect its conidiospores against the exogenous stress result-
ing from high temperatures and an oxidative
environment.2 The ability of mannitol to scavenge reac-
tive oxygen species (ROS) is exploited by the human
pathogen Cryptococcus neoformans and the tobacco
pathogen Alternaria alternata to suppress the host de-
fense strategies that are based on the generation of
ROS against the microbial parasite.3,4 The wheat path-
ogen Stagonospora nodorum requires mannitol for asex-
ual sporulation.5 The human pathogen Aspergillus
fumigatus, which is the most common etiologic agent
D-Mannitol is one of the most abundant sugar alcohols
in nature and ubiquitous throughout the fungal king-
dom. Recent evidence supporting a role of mannitol as
stress metabolite in the parasitic lifestyle of different fun-
gi has rekindled the interest in the physiological func-
tions fulfilled by mannitol in lower and higher
*
Corresponding authors. Tel.: +43 316 873 8400; fax: +43 316 873
8434 (B.N.); e-mail: bernd.nidetzky@TUGraz.at
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.04.011

S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
1415
for invasive aspergillosis in immunosuppressed hosts,
produces and releases sufficient amounts of mannitol
to raise serum mannitol levels of infected animals.6,7
Therefore, these findings suggest that the metabolism
of mannitol might be a viable target for the development
of novel antifungal strategies. The primary biosynthetic
route towards mannitol in Aspergilli and other fungi is
the reduction of D-fructose 6-phosphate (Fru6P) to D-
mannitol 1-phosphate (Man-ol1P) catalyzed by manni-
tol-1-phosphate 5-dehydrogenase (M1PDH). Man-
ol1P is subsequently dephosphorylated to mannitol.
According to similarity at the level of the amino acid
sequence, almost all the known fungal M1PDHs are
classified as polyol-specific long-chain dehydrogenases
and reductases (PSLDRs).8 The PSLDRs constitute a
distinct evolutionary lineage of NAD(P)+-dependent
secondary alcohol dehydrogenases. These do not require
a metal cofactor such as Zn2+ for catalysis and are usu-
ally active as monomers.9 The molecular size of
PSLDRs varies between 380 and 550 residues, whereby
M1PDHs constitute the smallest members of the family.
Structure-based sequence analysis has revealed that the
currently classified PSLDRs share a common structural
organization where the active site is located in a cleft
formed by an N-terminal Rossmann-fold coenzyme
binding domain and a largely a-helical domain that pro-
vides key elements of substrate binding recognition and
catalysis. With the exception of the early studies of the
enzyme from A. niger, little is currently known about
the relationships of structure and function of M1PDHs
from fungi.10
inhibition of the microbial parasite should be selective
considering that there are no PSLDR orthologues in
the human genome. Detailed study of AfM1PDH is
therefore not only of physiological interest but could
also eventually gain a clinical importance.
The analysis of the primary kinetic isotope effect
(KIE) resulting from the deuteration of the substrate
has provided a valuable insight into the kinetic and cat-
alytic mechanisms utilized by several NAD(P)+-depen-
dent dehydrogenases.11 The application of a similar
KIE strategy to examine AfM1PDH requires the synthe-
sis of protio and deuterio forms of Man-ol1P. We de-
scribe an enzymatic method (Scheme 1) that allows the
preparation of both substrates in high purity and yield.
To our knowledge, 5-[2H]-Man-ol1P has not been
reported in the literature so far.
2. Results and discussion
2.1. Cloning and expression of a putative M1PDH from
A. fumigatus
The whole genome shotgun sequence of A. fumigatus
shows an open-reading frame that encodes a putative
M1PDH (UniProt/TrEMBL entry Q4X1A4).12 Because
the coding region of the AfM1PDH gene is not inter-
rupted by introns, it was possible to amplify the entire
gene directly from genomic DNA of A. fumigatus. The
PCR product thus obtained had the expected size of
1187 bp and was cloned into a pQE-70 plasmid expres-
sion vector. The recombinant protein contained at its
N-terminus an additional leucine after the initiator
methionine that was introduced with the oligonucleotide
primer used for PCR. Furthermore, a peptide of eight
amino acids (-Arg-Ser-His6) was fused to the C-terminal
end of the protein to facilitate purification. Heterologous
In this paper, we report on the biochemical character-
ization of a recombinant M1PDH from the human
pathogenic mold A. fumigatus (AfM1PDH, EC
1.1.1.17). Assuming that mannitol constitutes a relevant
pathogenic factor of A. fumigatus, the biosynthetic
M1PDH is a potential drug target. M1PDH-directed
CH2OH
O
CH2O P
CH2O P
HO
HO
H
H
H
HO
HO
H
H
H
HO
H
H
or
Af M1PDH
OH
OH
OH
OH
CH2OH
OH
OH
CH2OH
H
H
2H
CH2O P
NAD+
+ H+
NADH/
[²H]-NADH
CO2
NaHCO2 or K[²H]CO2
CbFDH
Scheme 1. Enzymatic synthesis of Man-ol1P and 5-[2H]-Man-ol1P using the coupled enzyme system AfM1PDH and Candida boidinii formate
dehydrogenase (CbFDH).

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S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
expression of the AfM1PDH gene was carried out
in E. coli JM109. SDS PAGE of crude bacterial cell ex-
tracts revealed an abundant protein band at the position
corresponding to the expected molecular mass of
ꢀ44 kDa for the recombinant protein (data not shown).
The comparison of specific M1PDH activities in the cell
extract (80 U/mg) and the purified enzyme (170 U/mg)
indicated that the AfM1PDH accounted for about
50% of the total intracellular protein in E. coli.
2.2. Purification, and molecular and kinetic character-
ization of AfM1PDH
Figure 1. SDS PAGE of purified AfM1PDH: (A) AfM1PDH purified
by metal affinity chromatography; (B) Low molecular weight standard
(GE Healthcare).
AfM1PDH was purified to apparent homogeneity by a
single step of column chromatography, using copper-
loaded Chelating Sepharose. A balance of the purifica-
tion with respect to protein and enzyme activity is given
in Table 1. The isolated enzyme migrated as a single pro-
tein band in SDS PAGE (Fig. 1). In size exclusion chro-
matography, it was eluted in a single peak that
corresponded to the molecular mass of about 40 kDa
and contained all the applied protein and enzyme activ-
ity. Therefore, this result suggests that AfM1PDH is a
functional monomer, like other PSLDRs that have been
characterized at the protein level.
Table 2. Kinetic constants for purified AfM1PDH at 25 °C
Ksubstrate (mM)
Kcofactor (mM)
kcat (sꢂ1
8.5 0.4
125
)
Man-ol1P
Fru6P
0.23 0.02
2.1 0.2
0.75 0.09
0.016 0.001
3
Man-ol1P oxidation and Fru6P reduction were measured in 100 mM
Tris/HCl buffer, pH 7.1. See Section 3 for the concentrations of sub-
strates and coenzymes used.
number (kcat) for the direction of Fru6P reduction was
about 10 times that for the direction of Man-ol1P oxida-
tion. The Km value for NADH was less than one tenth
that for NAD+. By contrast, apparent binding affinity
(1/Km) for Man-ol1P was higher than that for Fru6P.
The catalytic efficiencies (kcat/Km) for reactions
with Fru6P (5.9 ꢁ 104 Mꢂ1 sꢂ1) and Man-ol1P (3.8 ꢁ
104 Mꢂ1 sꢂ1) in the presence of a saturating concentra-
tion of coenzyme were therefore in the same range.
Kinetic data obtained with AfM1PDH are well com-
parable with those for the M1PDH from A. niger re-
ported by Kiser and Niehaus.10 Like AfM1PDH, the
enzyme from A. niger shows a large preference
(P1000-fold) for the reaction with NAD(H) as com-
pared to NADP(H). The Km values for substrates and
coenzymes are similar for both fungal M1PDHs. The
M1PDH from Escherichia coli is also a monomer in
solution and like the fungal enzymes, it has a higher
affinity for NADH than for NAD+ at neutral pH.13 It
prefers NAD+ more than 100-fold over NADP+, and
its activity appeared to be independent of a metal
cofactor.14
The purified AfM1PDH displayed the predicted enzy-
matic function. It showed
a specific activity of
169 4 U/mg for the reduction of Fru6P by NADH
at pH 7.1. In the direction of NAD+-dependent oxida-
tion of Man-ol1P (100 mM glycine/NaOH buffer, pH
10.0), the specific activity was 17.6 1.4 U/mg. Note
that the pH values of measurement were chosen in a
suitable pH range for mannitol oxidation and fructose
reduction (data not shown). Using the same reaction
conditions, the specific enzyme activities for Fru6P
reduction by 0.2 mM NADPH and Man-ol1P oxidation
by 2.0 mM NADP+ were 0.58 0.05 U/mg and
0.08 0.01 U/mg, respectively. However, it must be
emphasized that AfM1PDH could not be saturated with
reduced and oxidized phosphorylated coenzyme in the
steady state, suggesting that the presence of the 20-phos-
phate of NADP(H) strongly decreased the binding affin-
ity for the coenzyme.
Michaelis–Menten constants for the reduction of
Fru6P by NADH and the oxidation of Man-ol1P by
NAD+ were determined at neutral pH and are summa-
rized in Table 2. Under the conditions used, the turnover
Table 1. Purification of recombinant AfM1PDH expressed in E. coli
Purification stage
Total activity (U)
Protein (mg/mL)
Specific activity (U/mg)
Total yield (%)
Purification factor
Crude extract
Affinity chromatography
Desalting
28000
24000
21600
50
4
11
80
135
169
100
86
77
1
1.7
2.1
Results are based on processing 2 g moist bacterial biomass. The AfM1PDH activity was assayed in the direction of Fru6P reduction at 25 °C using
100 mM Tris/HCl buffer, pH 7.1. The concentrations of Fru6P and NADH were 50 mM and 0.2 mM, respectively.

S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
1417
Figure 2. Enzymatic synthesis of Man-ol1P monitored by HPAE-PAD: (A) Dashed line: Superimposed traces of authentic standards of Man-ol1P
(13.7 min), Glc6P (16.7 min) and Fru6P (18.4 min); Solid lines: Chromatograms for the first 210 min of Man-ol1P production from Fru6P using
CbFDH fraction I for cofactor recycling; (B) time course of Man-ol1P production from 200 mM Fru6P. The solid line indicates the trend of the data.
Membership of AfM1PDH to the PSLDR protein
family leads to the suggestion that the catalytic activity
of the enzyme is not dependent on a metal cofactor.
To verify the structure-derived implication, we incu-
bated purified AfM1PDH (0.27 lg/mL) in the presence
of 100 mM EDTA for 1 h at room temperature and as-
sayed at various times the residual activity of the enzyme
in the direction of Fru6P reduction. No loss of activity
was observed relative to a control in which EDTA was
lacking, as expected for a metal-independent enzyme.
NADH is achieved via NAD+-dependent oxidation of
formate into CO2 catalyzed by CbFDH.
A typical time course of synthesis of Man-ol1P by the
coupled action of dehydrogenases is summarized in Fig-
ure 2. We used a 2.5-fold excess of CbFDH activity
(5 U/mL) over AfM1PDH activity (2 U/mL) to ensure
that a substantial portion of the total coenzyme, which
was added as NAD+ at the start of the reaction
(0.5 mM), was present in the required reduced form.
Furthermore,
a
slight molar excess of formate
(250 mM) over Fru6P (200 mM) was chosen to ensure
the complete conversion of ketose substrate into prod-
uct. A partially purified preparation of CbFDH with a
specific activity of 1.4 U/mg was used in the reaction.
High performance anion exchange chromatography
with pulsed amperometric detection (HPAE-PAD) was
employed for monitoring the progress of the reaction,
as shown in panel A of Figure 2. Under the conditions
used, more than 99% of the initial Fru6P was converted
into Man-ol1P. Panel B of Figure 2 shows the time
course of Man-ol1P production. The workup of Man-
ol1P included the removal of the enzymes by ultrafiltra-
tion as the first step. Further purification targeted the
elimination of NAD(H) because the contamination of
the product with coenzyme could interfere with the
planned enzyme kinetic measurements. Because anion
exchange chromatography on a MonoQ 5/50 GL (GE
Healthcare) column was not successful, we focused on
selective precipitation of Man-ol1P as barium salt. A
screening of conditions for the recovery of product in
the highest possible purity revealed that the addition
of an equal volume of EtOH to the reaction mixture
facilitated precipitation of Man-ol1P while NAD(H)
and residual formate were retained in the supernatant.
After washing and drying the precipitate, Man-ol1P
was obtained in an isolated yield of 90% and a purity
2.3. Enzymatic synthesis of D-mannitol 1-phosphate and
5-[2H]-D-mannitol 1-phosphate
Previously reported routes for the chemical synthesis of
Man-ol1P started from mannose 6-phosphate or manni-
tol.10,15,16 A major drawback of using mannose 6-phos-
phate is the high cost of the substrate. Phosphorylation
of mannitol was performed with phosphorylchloride in
pyridinic solution. It required harsh reaction conditions
and consumed large amounts of organic solvents. A het-
erogeneous mixture of mannitol-phosphates was ob-
tained from which 90% pure Man-ol1P was recovered
through a laborious re-crystallization procedure. The
overall yield was therefore only ꢀ9%.17 None of the de-
scribed methods provide a direct access to the deuter-
ated analogue of Man-ol1P.
We therefore designed a new synthetic route that is
summarized in Scheme 1 and can be flexibly applied
for the preparation of Man-ol1P or 5-[2H]-Man-ol1P.
A bi-enzymatic system constituted of AfM1PDH and
formate dehydrogenase from Candida boidinii (CbFDH)
was employed. AfM1PDH catalyzes the desired syn-
thetic reaction transforming Fru6P into Man-ol1P via
NADH-dependent reduction, and the regeneration of

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S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
Man-ol1P without affecting negatively the space-time
yield of the reaction as well as the yield and purity of
the final product.
Substitution of formate by [2H]-formate as reductant
for the enzymatic conversion of Fru6P was expected
to provide convenient access to the deuterated analogue
of Man-ol1P, namely 5-[2H]-D-mannitol 1-phosphate (5-
[2H]-Man-ol1P). Using reaction conditions otherwise
identical to those for Man-ol1P production, we ob-
served the complete conversion of ketose substrate but
only partial (ꢀ2/3) deuteration of position C–5 in the
isolated Man-ol1P product. Because the deuterium label
in the commercial [2H]-formate had been verified by
NMR spectroscopy. We examined rigorously the role
of the purity of CbFDH, using for 5-[2H]-Man-ol1P syn-
thesis each of the different enzyme preparations shown
in Table 3. The highly purified CbFDH obtained after
anion exchange chromatography (Fig. 4) was required
to synthesize 5-[2H]-Man-ol1P with a high degree of
deuterium labeling. The isotopic purity of the isolated
product was estimated by NMR to be >95% 2H
(Fig. 3). To our knowledge, the synthesis of 5-[2H]-
Man-ol1P has not been reported so far.
Figure 3. 13C spectra of D-5-[2H]-mannitol 1-phosphate (A) and D-
mannitol 1-phosphate (B).
of >95% (determined by HPAE-PAD and NMR). Using
NMR analysis, neither NAD+ nor NADH could be de-
tected in the isolated product. A possible contamination
of the product by Fru6P, D-glucose 6-phosphate
(Glc6P), formate, mannitol, and fructose was ruled
out within the limits of detection of the used HPAE-
PAD and NMR methods. A 13C NMR spectrum of
the final product is shown in Figure 3.
We asked the question of whether a causal relationship
exists between the removal of PGI activity from the
CbFDH preparation and the enhancement of deuterium
label in the 5-[2H]-Man-ol1P product. A deuterium
Inspection of the HPAE-PAD traces in Figure 2A
shows that immediately after the start of the reaction,
a new compound was formed which according to its
retention time (16.7 min) could be clearly distinguished
from Man-ol1P and was identified as Glc6P. The Glc6P
gradually disappeared as the production of Man-ol1P
progressed. Because no Glc6P was added with the sub-
strate, the results imply the presence of an isomerase
activity that reversibly interconverts Fru6P and Glc6P.
The only possible source for this activity was the par-
tially purified CbFDH. We therefore measured the activ-
ity of phosphoglucose isomerase (PGI) in the used
preparation of CbFDH and the E. coli cell extract from
which it was obtained. The results summarized in Table
3 show that the heat treatment employed in the purifica-
tion of CbFDH only partially eliminated the PGI activ-
ity present in the starting material. A useful consequence
of the PGI activity carried over with the CbFDH prep-
aration was that Glc6P, which is cheaper than Fru6P,
could be employed as a substrate for the synthesis of
Figure 4. CbFDH purification documented by SDS PAGE analysis:
(A) low molecular weight standard (GE Healthcare); (B) purified
CbFDH obtained after anion exchange chromatography (fraction III);
(C) preparation after heat treatment and desalting (fraction II); (D)
preparation after heat treatment (fraction I); (E) crude E. coli cell
extract.
Table 3. Purification of recombinant CbFDH
Purification stage
Total activity Protein
Specific activity Total yield Purification factor FDH Total activity Specific activity
FDH (U)
(mg/mL) FDH (U/mg)
FDH (%)
PGI (U)
PGI (U/mg)
Crude extract
Heat denaturation 185
Desalting
Q-Sepharose FF
249
40.6
6.4
9.4
1.0
1.4
1.4
4.4
100
74
62
1.0
1.5
1.5
4.5
317
232
203
<0.06
1.2
1.8
1.9
153
131
5.22
53
<0.002
Results are based on processing 3.3 g wet cells of E. coli. Desalting was performed by separating the total amount of protein in two batches which was
necessary due to the high concentration of formate carried over from the heat treatment. The activities of CbFDH and PGI were assayed as described
under Section3.

S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
1419
wash-out experiment was performed in which the com-
pletely deuterated 5-[2H]-Man-ol1P (75 mM) was incu-
bated at room temperature and pH 7.5 in the presence
of the PGI-containing standard preparation of CbFDH
(obtained after heat treatment) or a commercial PGI
from baker’s yeast. Incubation with 2.3 U PGI/mL was
done for 22 h and the product was isolated as described
above. No deuterium wash-out was observed within the
limits of experimental error of 13C NMR analysis. Fur-
ther examination of the interesting reaction that leads
to partial deuteration of Man-ol1P was beyond the remit
of this study and is left for consideration in the future.
bacter aerogenes and E. coli.9,18,19 The stereoselectivity
of biocatalytic hydrogen transfer to NAD+ reflects the
relative orientation of substrate and coenzyme upon
their binding to the enzyme. It is dictated by the protein
structure and therefore expected to be conserved among
evolutionary related dehydrogenases. The pro-S stereo-
selectivity of AfM1PDH is consistent with the proposed
membership of the enzyme to the family of
PSLDRs.20,21
2.5. KIE on NAD+-dependent enzymatic oxidation of
D-mannitol 1-phosphate
2.4. Stereochemical course of hydrogen transfer catalyzed
by AfM1PDH
Figure 6 compares time courses of the enzymatic oxida-
tion of Man-ol1P and 5-[2H]-Man-ol1P obtained under
conditions in which the concentrations of the substrate
(6 mM) and NAD+ (6 mM) were saturating in the stea-
dy state and therefore maximum initial rates (Vmax) are
measured. It shows that substrate deuteration caused a
significant slowing down of the reaction catalyzed by
AfM1PDH. Because the molar enzyme concentration
was identical in the two experiments, the slopes of linear
plots of the concentration of reduced coenzyme against
the reaction time could be directly used to calculate the
KIE, which is the ratio of the slopes measured with pro-
tio and deuterio substrate and had a value of 2.9 0.2.
The degree of rate reduction expressed in the value of
KIE depends on how much the isotope-sensitive step
of hydride transfer contributes to the overall reaction.
Under the Vmax conditions used, the substrate binding
steps are not relevant kinetically. However, microscopic
reaction steps comprised in the value of Vmax include ki-
netic isomerizations of substrate-bound enzyme forms,
the chemical conversion steps, and the release of product
and NADH to regenerate the free enzyme. The dissoci-
ation of the NAD(P)H product constitutes the rate-
determining step in reactions catalyzed by many
NAD(P)+-dependent dehydrogenases are usually highly
stereoselective with regard to transfer of hydrogen from
the prochiral C–4 of the nicotinamide moiety of NADH
or NADPH. These are classified according to stereo-
chemical preference as pro-R or pro-S specific. To deter-
mine the stereochemical course of the reaction catalyzed
by AfM1PDH, we incubated the purified enzyme in the
presence of 5-[2H]-Man-ol1P and NAD+ and monitored
1
by using in situ H NMR analysis the formation of the
deuterated, hence chiral NADH product. The results in
Figure 5 reveal that (4S)-[2H]-NADH was produced
upon the enzymatic oxidation of 5-[2H]-Man-ol1P.
The stereoselectivity of the reduction of NAD+ was
absolute within the limits of detection of the chosen ana-
lytical method. Therefore, these results imply that the
AfM1PDH belongs to the group of pro-S specific dehy-
drogenases, a functional classification that the fungal
enzyme shares with M1PDHs from the bacteria Aero-
Figure 5. Stereospecificity of hydrogen transfer to the nicotinamide
moiety of NAD(H) catalyzed by AfM1PDH: (A) 5-[2H]-Man-ol1P and
NAD+ before enzymatic conversion; (B) formation of (4S)-[2H]-
NADH after enzymatic reaction for 30 min; incubations in (A) and (B)
were carried out using 2 mM NAD+, 5 mM 5-[2H]-Man-ol1P in
30 mM Tris/HCl, p2H 7.1; (C) formation of NADH and (4S)-[2H]-
NADH after 15 min of enzymatic reaction using 5 mM NAD+ and
5 mM of a 2:1 mixture of 5-[2H]-Man-ol1P/Man-ol1P in 50 mM Tris/
HCl, p2H 9.8.
Figure 6. Primary deuterium isotope effect on kcat of Man-ol1P/5-[2H]-
Man-ol1P oxidation: initial rates of Man-ol1P oxidation (d) and 5-
[2H]-Man-ol1P oxidation (s). The concentrations of substrate and
NAD+ were 6 mM each.

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S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
NAD(P)+-dependent dehydrogenases, reflected by a
KIE on Vmax that is equal to or not much larger than
unity.11 In AfM1PDH, by contrast, the chemical step
of hydride transfer strongly governs the maximum rate
of Man-ol1P oxidation by NAD+ at pH 7.1.
Summarizing, we have functionally expressed the
M1PDH from A. fumigatus in E. coli and performed a
biochemical characterization of the recombinant en-
zyme. A new method of synthesis of Man-ol1P and its
deuterio analogue 5-[2H]-Man-ol1P was established.
Using the KIE approach thus made possible, we deter-
mined that hydride transfer to NAD+ is a slow step in
the overall oxidation of Man-ol1P and product release
is not rate-determining.
7.5 was chosen because both AfM1PDH and CbFDH
show good activity and stability under these conditions.
After the reaction, enzymes were removed by ultrafiltra-
tion using Vivaspin 6 MWCO 10 kDa microconcentrator
tubes (Sartorius AG, Goettingen, Germany) at 4500 g.
Man-ol1P was precipitated through the addition of an
equimolar amount of BaSO4 followed by the dilution of
the reaction mixture with the same volume of absolute
EtOH. Precipitation was allowed to proceed for 2 h at
4 °C. The precipitate was collected by filtration and
washed exhaustively with absolute EtOH. The barium
salt of Man-ol1P was dried over night at 40 °C. 1H
NMR (D2O): ppm 3.81 (m, 2H), 3.71 (m, 2H), 3.63 (m,
3H), 3.49 (dd, 1H, J1 11.7 Hz, J2 5.3 Hz); 13C NMR
(D2O): ppm 71.0 (C-5), 70.3 (C-1, d, JC–P 5.8 Hz), 69.4
(C-4), 68.5 (C-3), 65.4 (C-2, d, JC–P 4.8 Hz), 63.4 (C-6).
The isolated product showed the same retention time in
HPAE-PAD (13.7 min) as the authentic Man-ol1P stan-
dard obtained from Sigma–Aldrich.
3. Experimental
3.1. Materials
Fructose 6-phosphate (Fru6P), D-mannitol 1-phosphate
(Man-ol1P), b-nicotinamide adenine dinucleotides
(NAD(P)+, NAD(P)H), sodium formate, potassium
[2H]-formate, salts and buffer reagents were from Sig-
ma–Aldrich (St. Louis, MO, USA). T4-Ligase, restric-
tion enzymes and Taq DNA polymerases were from
Fermentas (Burlington, Canada). The pQE70-vector
was obtained from Qiagen (Hilden, Germany). PWO-
polymerase was a product of PEQLAB (Erlangen, Ger-
many). Oligonucleotides were obtained from VBC-Bio-
tech (Vienna, Austria). Genomic DNA of A. fumigatus
was kindly provided by Dr. Hubertus Haas (Division
of Molecular Biology, Medical University of Innsbruck,
Austria).
3.3. 5-[2H]-D-Mannitol 1-phosphate
The synthesis of 5-[2H]-Man-ol1P used the analogous
procedure employed for the production of Man-ol1P ex-
cept that potassium [2H]-formate served as the reductant
and a purified preparation of CbFDH (fraction III; see
1
Section 3.6) was utilized. H NMR (D2O): ppm 3.81
(m, 2H), 3.71 (m, 2H), 3.63 (m, 2H), 3.49 (d, 1H, J
11.7 Hz); 13C NMR (D2O): 70.6 (C-5, t, JC–D 22.5 Hz),
70.3 (C-1, d, JC–P 5.8 Hz), 69.3 (C-4), 68.6 (C-3), 65.4
(C-2, d, JC–P 4.8 Hz), 63.3 (C-6). The 5-[2H]-Man-ol1P
eluted in HPAE-PAD at the same retention time
(13.7 min) as the authentic Man-ol1P standard.
3.4. Analytical methods
3.2. D-Mannitol 1-phosphate
3.4.1. Protein analysis. Purification of CbFDH and
AfM1PDH was monitored by SDS PAGE using the
Phast System (GE Healthcare, Chalfont St. Giles, United
Kingdom). Staining of protein bands was done with
Coomassie Brilliant Blue. Size exclusion chromatogra-
phy of purified AfM1PDH was carried out on a Superdex
200 HR 10/30 gel filtration column (GE Healthcare). The
column was calibrated with a gel filtration standard from
Bio-Rad Laboratories (Hercules, USA).
Enzymatic conversion of Fru6P or Glc6P (200 mM) to
Man-ol1P was carried out in a 30 mM Tris/HCl buffer,
pH 7.5, containing 250 mM sodium formate, 0.5 mM
NAD+, 5 U/mL partially purified CbFDH (fraction I;
see Section 3.6), and 2 U/mL purified AfM1PDH. En-
zyme activities of CbFDH (NAD+-dependent oxidation
of formate) and AfM1PDH (NADH-dependent reduc-
tion of Fru6P) were measured at 25 °C under conditions
in which the concentrations of substrate and coenzyme
were saturating (CbFDH: 250 mM formate, 2 mM
NAD+; AfM1PDH: 50 mM Fru6P, 0.2 mM NADH).
The enzyme assays were performed in 30 mM Tris/
HCl, pH 7.5.
The synthesis of Man-ol1P was carried out in a batch
reaction at room temperature, typically for 20 h. The
working volume was 10 mL. The pH of the reaction was
controlled at a value of 7.5 through the addition of diluted
HCl. Gentle mixing with a magnetic stirrer was used be-
fore taking samples or while adjusting the pH. A pH of
3.4.2. Assays. Photometric measurements were carried
out with a DU800 spectrophotometer from Beckman
Coulter Inc. (Fullerton, CA, USA). The activities of
CbFDH and AfM1PDH were determined from initial
rate measurements in which the production or consump-
tion of NADH at
a
wavelength of 340 nm
(e = 6.22 cmꢂ1 mMꢂ1) was measured. Unless otherwise
indicated, the activity of AfM1PDH was assayed at
25 °C either in the direction of NADH-dependent reduc-
tion of Fru6P using a 100 mM Tris/HCl buffer, pH 7.1,

S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
1421
or in the direction of NAD+-dependent oxidation of
Man-ol1P using a 100 mM glycine/NaOH buffer, pH
10.0. The concentrations of Fru6P and NADH were
50 mM and 0.2 mM, respectively. The concentrations
of Man-ol1P and NAD+ were 0.5 mM and 2 mM,
respectively. The activity of CbFDH was assayed in
the direction of NAD+-dependent oxidation of formate
at 30 °C using 100 mM potassium phosphate buffer, pH
7.5. The concentrations of formate and NAD+ were
162 mM and 2 mM, respectively.
NMR spectra of Man-ol1P and 5-[2H]-Man-ol1P
15000 scans were accumulated and 1 Hz line broadening
was used for sensitivity enhancement prior to Fourier
transformation. Enzymatic conversions of NAD+
(2 mM or 5 mM) into [2H]-NADH and NADH with
AfM1PDH (ꢀ1 lM) using Man-ol1P/5-[2H]-Man-ol1P
(5 mM) in 30 mM Tris/HCl, p2H 7.1, or 50 mM Tris/
HCl, p2H 9.8, were performed directly in the NMR sam-
ple tube, which is placed in the magnet. Proton NMR
spectra were recorded by acquiring 30272 data points
and accumulation of 200 scans.
PGI activity was measured in a coupled enzymatic as-
say at 30 °C essentially as described by Ruijter and Vis-
ser except that AfM1PDH was used instead of the
M1PDH from A. nidulans.22 The principle of the assay
is that Glc6P is isomerised by PGI to Fru6P, which is
in turn reduced by AfM1PDH. Therefore, PGI activity
is measured as the decrease in the absorbance of NADH
at 340 nm. The assay was performed in 100 mM potas-
sium phosphate buffer, pH 7.5, and contained 20 mM
Glc6P, ꢀ4 U/mL AfM1PDH, and 0.2 mM NADH.
Protein concentrations were measured with the Bio-
Rad Protein Assay, which is based on the method of
Bradford and was calibrated against BSA.23 Man-ol1P
and 5-[2H]-Man-ol1P were quantified enzymatically
using AfM1PDH in 100 mM glycine/NaOH buffer, pH
10.0, containing 2 mM NAD+. Concentrations were cal-
culated from the formed NADH using appropriate cal-
ibration of the assay with authentic Man-ol1P in the
range of 0.04–0.20 mM.
3.5. Gene cloning, and production and purification of
recombinant AfM1PDH
The intron-less open-reading frame encoding
AfM1PDH was amplified from genomic DNA of A.
fumigatus Af293 using PCR with PWO-polymerase
and the following pair of oligonucleotide primers:
forward primer: 50-GATCTAGCATGCTAGGAAAG-
AAGGCTATCCAGTTTG-30,
reverse primer: 50-GATCTAAGATCTCTTGCTGTC-
CTTCTGCACCTT-30.12
The PCR product was digested with SphI and BglII
and inserted into the plasmid vector pQE70 previously
treated with the same restriction enzymes. The obtained
construct was transformed into E. coli JM109. Cells har-
boring pQE70-AfM1PDH were cultivated at 37 °C in
LB-Lennox-medium containing 115 mg/L ampicillin.
Expression of recombinant AfM1PDH was induced by
IPTG (125 lM) in mid-exponential growth phase after
the temperature was decreased to 25 °C. After about
20 h post-induction, E. coli biomass was harvested by
centrifugation (4400 g, 20 min, 4 °C) and disrupted twice
with a French Press at 1500 psi cell pressure. Debris of
bacterial cells were removed by ultracentrifugation at
80000 g for 45 min at 4 °C. The crude cell extract was
then applied on a column of copper-loaded Chelating
Sepharose Fast Flow (XK 16 column from GE Health-
care; 1.6 ꢁ 6.2 cm) equilibrated with a 50 mM Tris/HCl
buffer, pH 7.1, that contained 300 mM sodium chloride
and glycerol (10% v/v). Competitive elution was
achieved using a linear gradient of imidazole in the
range of 0–220 mM which was applied over eight col-
umn volumes while employing a constant flow rate of
1.5 mL/min. Imidazole, sodium chloride and glycerol
were afterwards removed using a HiPrep 26/10 pre-
packed desalting column (GE Healthcare).
3.4.3. Carbohydrate analysis. High performance anion
exchange chromatography with pulsed amperometric
detection (HPAE-PAD) was utilized to measure the con-
centration of Fru6P, Glc6P, and Man-ol1P or 5-[2H]-
Man-ol1P in samples taken from enzymatic conversions.
The analysis was performed using a Dionex BioLC sys-
tem (Dionex Corporation, Sunnyvale, CA, USA)
equipped with a CarboPac PA10 column (4 ꢁ 250 mm)
and an Amino Trap guard column (4 ꢁ 50 mm) thermo-
stated at 30 °C. Phosphorylated sugars were detected
with an ED50A electrochemical detector using a gold
working electrode and a silver/silver chloride reference
electrode by applying the predefined waveform for car-
bohydrates. Elution was carried out at a flow rate of
1 mL/min based on a previously described method with
a linear gradient from 50 to 275 mM NaOAc applied
within 22 min in an isocratic background of 4 mM
NaOH.24 Column was flushed 5 min with 800 mM NaO-
Ac (+4 mM NaOH) and 15 min with the starting condi-
tions before the next sample was injected.
3.4.4. NMR. Measurements were carried out on a Var-
ian INOVA 500 MHz spectrometer (Varian Inc., Palo
Alto, CA, USA) at 22 °C in D2O (99.9% D) using the
3.6. Gene cloning, and production and purification of
recombinant CbFDH
1
VNMR 6.1c software. H NMR spectra were measured
C. boidinii ATCC 18810 was grown on YPD medium
(10 g/L yeast extract, 20 g/L peptone and 20 g/L
at 499.98 MHz and 13C NMR at 125.69 MHz. For 13C

1422
S. Krahulec et al. / Carbohydrate Research 343 (2008) 1414–1423
glucose), and its genomic DNA was isolated using the
WizardÒ Genomic DNA Purification Kit from Promega
(Madison, WI, USA). The gene encoding CbFDH (EC
1.2.1.2) was amplified by PCR with the High Fidelity
PCR Enzyme Mix from Fermentas (Burlington, Can-
ada) and the following pair of oligonucletide primers:
parameters for NADH-dependent reduction of Fru6P
were obtained using concentrations of NADH between
0.002 and 0.2 mM or Fru6P between 0.5 and 40 mM,
while keeping the other substrate constant and saturat-
ing (45 mM Fru6P; 0.2 mM NADH). The enzyme was
appropriately diluted in the corresponding reaction buf-
fer. Reactions were started with enzyme and initial rates
were recorded photometrical as described in Section 3.4.
Steady state kinetic parameters were determined by fit-
ting the Michaelis–Menten equation to the experimental
data using Sigma Plot 9.0 (SYSTAT Software Inc., San
forward primer: 50-GATCTAGAATTCATGAAGAT-
CGTTTTAGTCTTATATGATGCTG-30,
reverse primer: 50-GATCTACTGCAGTTATTTCTTA-
TCGTGTTTACCGTAAGCTTTAG-30.
Jose, CA, USA). The kinetic turnover numbers (kcat
)
The PCR product was digested with EcoRI and PstI
and inserted into the plasmid vector pBTac1 (Boehrin-
ger Mannheim, Germany) previously cleaved with the
same restriction enzymes. Identity of the primary amino
acid sequence with the CbFDH described by Sakai et al.
(UniProt/TrEMBL entry O93968) was confirmed by
DNA sequencing.25 Expression of recombinant CbFDH
was done in E. coli JM109, using the procedure de-
scribed under Section 3.5. Purification of CbFDH was
performed using slight modifications of the protocol de-
scribed by Slusarczyk et al. for the isolation of FDH1
from C. boidinii (UniProt/TrEMBL entry O13437).26
Briefly, the crude E. coli extract (obtained as described
under 3.5) was incubated in the presence of 10% w/v for-
mate or [2H]-formate at 60 °C for 5 min. Precipitated
proteins were removed by centrifugation (11200 g,
10 min, 4 °C). The supernatant (fraction I) was desalted
in two batches using a pre-packed desalting column (see
Section 3.5). The desalted protein solution (fraction II)
was loaded on a column of Q-Sepharose Fast Flow
(XK 16 column from GE Healthcare; 1.6 ꢁ 9.0 cm)
equilibrated with 10 mM potassium phosphate buffer,
pH 7.5. At a flow of 10 mL/min the column was flushed
with 4.5 column volumes of the phosphate buffer before
adding up to 150 mM NaCl in a linear gradient of 50
column volumes. Purified CbFDH was stabilized by
the addition of either 200 mM sodium formate or potas-
sium [2H]-formate (fraction III).
were calculated from the maximal initial velocities using
the molecular mass of 44.2 kDa as derived from the
unprocessed amino acid sequence of recombinant
AfM1PDH. Primary deuterium KIE on kcat in the direc-
tion of alcohol oxidation was obtained by using saturat-
ing concentration of Man-ol1P or 5-[2H]-Man-ol1P
(6 mM) and NAD+ (6 mM), employing otherwise iden-
tical reaction conditions as described above for Man-
ol1P oxidation.
Acknowledgments
We are especially grateful to Dr. Hubertus Haas (Divi-
sion of Molecular Biology, Medical University of Inns-
bruck, Austria) for providing genomic DNA of A.
fumigatus. We thank Eva Glaumann for assistance in
cloning of CbFDH. Financial support from the Aus-
trian Science Fund FWF (P18275-B09) is gratefully
acknowledged.
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