Báo cáo khoa học: Enzymes of mannitol metabolism in the human pathogenic fungusAspergillus fumigatus– kinetic properties of mannitol-1-phosphate 5-dehydrogenase and mannitol 2-dehydrogenase, and their physiological implications pot

Enzymes of mannitol metabolism in the human pathogenic
fungus Aspergillus fumigatus – kinetic properties of
mannitol-1-phosphate 5-dehydrogenase and mannitol
2-dehydrogenase, and their physiological implications
Stefan Krahulec, Guilliano Cem Armao, Mario Klimacek and Bernd Nidetzky
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria
Keywords
Aspergillus fumigatus; mannitol; mannitol
metabolism; mannitol-1-phosphate 5-
dehydrogenase; mannitol 2-dehydrogenase
Correspondence
B. Nidetzky, Institute of Biotechnology and
Biochemical Engineering, Graz University of
Technology, Petersgasse 12 ⁄ I, A-8010 Graz,
Austria
Fax: +43 316 873 8434
Tel: +43 316 873 8400
E-mail:
(Received 17 December 2010, revised 1
February 2011, accepted 4 February 2011)
doi:10.1111/j.1742-4658.2011.08047.x
The human pathogenic fungus Aspergillus fumigatus accumulates large
amounts of intracellular mannitol to enhance its resistance against defense
strategies of the infected host. To explore their currently unknown roles in
mannitol metabolism, we studied A. fumigatus mannitol-1-phosphate
5-dehydrogenase (AfM1PDH) and mannitol 2-dehydrogenase (AfM2DH),
each recombinantly produced in Escherichia coli, and performed a detailed
steady-state kinetic characterization of the two enzymes at 25 °C and
pH 7.1. Primary kinetic isotope effects resulting from deuteration of alco-
hol substrate or NADH showed that, for AfM1PDH, binding of
D-manni-

tol 1-phosphate and NAD
+
is random, whereas D-fructose 6-phosphate
binds only after NADH has bound to the enzyme. Binding of substrate
and NAD(H) by AfM2DH is random for both
D-mannitol oxidation and
D-fructose reduction. Hydride transfer is rate-determining for D-mannitol
1-phosphate oxidation by AfM1PDH (k
cat
= 10.6 s
)1
) as well as D-fructose
reduction by AfM2DH (k
cat
=94s
)1
). Product release steps control the
maximum rates in the other direction of the two enzymatic reactions. Free
energy profiles for the enzymatic reaction under physiological boundary
conditions suggest that AfM1PDH primarily functions as a
D-fructose-6-
phosphate reductase, whereas Af M2DH acts in
D-mannitol oxidation, thus
establishing distinct routes for production and mobilization of mannitol in
A. fumigatus. ATP, ADP and AMP do not affect the activity of
AfM1PDH, suggesting the absence of flux control by cellular energy charge
at the level of
D-fructose 6-phosphate reduction. AfM1PDH is remarkably
resistant to inactivation by heat (half-life at 40 °C of 20 h), consistent with
the idea that formation of mannitol is an essential component of the tem-

perature stress response of A. fumigatus. Inhibition of AfM1PDH might be
a useful target for therapy of A. fumigatus infections.
Abbreviations
AbM2DH, mannitol 2-dehydrogenase from Agaricus bisporus; AfM1PDH, mannitol-1-phosphate 5-dehydrogenase from
Aspergillus fumigatus; AfM2DH, mannitol 2-dehydrogenase from Aspergillus fumigatus; EcM1PDH, mannitol-1-phosphate 5-dehydrogenase
from Escherichia coli; Fru,
D-fructose; Fru6P, D-fructose 6-phosphate; KIE, kinetic isotope effect; Man-ol, D-mannitol; Man-ol1P, D-mannitol
1-phosphate; M1PDH, mannitol-1-phosphate 5-dehydrogenase; M2DH, mannitol 2-dehydrogenase; NADD, (4S)-[
2
H]-NADH; PSLDR, polyol-
specific long-chain dehydrogenase ⁄ reductase; PsM2DH, mannitol 2-dehydrogenase from Pseudomonas fluorescens.
1264 FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS
Introduction
The six-carbon polyol d-mannitol (Man-ol) is ubiqui-
tous throughout the fungal kingdom, and one of the
most abundant sugar alcohols in nature. Aside from
other physiological functions that have been ascribed
to it, intracellular accumulation of Man-ol is a wide-
spread mechanism by which fungi cope with different
forms of external stress, including high temperature
and oxidative stress [1]. In parasitic fungi, the
improved stress resistance resulting from accumulated
Man-ol appears to confer a substantially enhanced
ability to deal with defense strategies of the infected
host [2–6]. The human pathogen Aspergillus fumigatus,
which is the most common agent causing invasive
aspergillosis in immunosuppressed patients, produces
enough Man-ol to raise the serum Man-ol level of the
infected animal [7,8]. High thermotolerance is a major
component of A. fumigatus pathogenicity, and involves

Man-ol indirectly [9]. Decreased susceptibility to reac-
tive oxygen species produced by human phagocytes in
response to the microbial infection is yet another viru-
lence factor of A. fumigatus, and includes direct partic-
ipation of Man-ol as a free radical scavenger [10,11].
The inhibition of fungal Man-ol production therefore
presents a potential target in advanced therapies for
A. fumigatus infections. Unfortunately, little is cur-
rently known about the enzyme system of Man-ol
metabolism in A. fumigatus.
A number of studies suggest that two different meta-
bolic paths contribute to the biosynthesis of Man-ol in
fungi (Scheme 1; reviewed in [1]). Reduction of d-fruc-
tose 6-phosphate (Fru6P) by an NADH-dependent
mannitol-1-phosphate 5-dehydrogenase (M1PDH; EC
1.1.1.17) gives d-mannitol 1-phosphate (Man-ol1P),
which, upon hydrolysis of phosphate ester by a phos-
phatase (EC 3.1.3.22), yields Man-ol (path 1). In the
alternative route (path 2), Fru6P is converted to
d-fructose (Fru), which, in turn, is reduced to Man-ol
by NADPH-dependent or NADH-dependent mannitol
2-dehydrogenase (M2DH; EC 1.1.1.138 or 1.1.1.67).
Mobilization of Man-ol occurs through quasireversal
of path 2, whereby an NADP
+
-dependent or NAD
+
-
dependent M2DH produces Fru, which is then phos-
phorylated from ATP by hexokinase (EC 2.7.1.1) to

regenerate Fru6P [12]. It is currently not clear whether
stockpiled mannitol could also be utilized by going
through the steps of path 1 in reverse [13–16], whereby
Man-ol would have to become phosphorylated by a
suitable kinase.
M1PDH and NAD
+
-dependent M2DH have
recently been characterized from A. fumigatus [17,18].
On the basis of sequence similarity, both enzymes were
classified as members of the polyol-specific long-chain
dehydrogenase ⁄ reductase (PSLDR) family [12]. The
PSLDRs constitute a diverse group of NAD(P)-
dependent oxidoreductases that are widespread among
microorganisms but are lacking in humans. Their acti-
vity is not dependent on a metal cofactor and is exclu-
sively targeted towards polyol ⁄ ketose substrates [12,19].
In a family-wide categorization of the PSLDRs,
M1PDH and M2DH were clearly separated from each
other, showing only a distant evolutionary relationship
[12,19]. Structurally, PSLDRs are composed of two
separate domains. The N-terminal domain adopts an
expanded Rossmann fold, and provides the residues for
binding the coenzyme. The C-terminal domain has a lar-
gely a-helical structure, and serves mainly in substrate
binding. The active site is located in the interdomain
cleft, and contains a highly conserved tetrad of residues
(Lys ⁄ Asn ⁄ Asn ⁄ His), whereby Lys is the catalytic acid–
base of the enzymatic reaction [20,21]. A recent study
has demonstrated, at the level of both gene transcript

and translated protein, that A. fumigatus M1PDH
(AfM1PDH) becomes strongly upregulated during
heat shock [9]. Enhanced biosynthesis of Man-ol via
AfM1PDH-catalyzed conversion of Fru6P might
contribute extra robustness to A. fumigatus under high-
temperature conditions.
In this study, an enzymological approach was chosen
to examine the roles of AfM1PDH and A. fumigatus
M2DH (AfM2DH) in the metabolism of Man-ol in
A. fumigatus. A detailed steady-state kinetic character-
ization was performed with each enzyme, providing the
basis for the construction of free energy profiles for the
enzymatic reactions under physiological boundary con-
ditions as defined from the literature. The results pro-
vide clear assignment of a biosynthetic function to
AfM1PDH (path 1), which behaves kinetically as a
Fru6P reductase, whereas AfM2DH is essentially a
Man-ol-oxidizing enzyme (path 2, backwards). The
results of inhibition studies show that the activity of
AfM1PDH would not be affected by changes in the lev-
els of ATP, ADP and AMP, suggesting that flux
Scheme 1. Metabolic pathways for the biosynthesis of mannitol in
fungi. HXK, hexokinase; M1Pase, mannitol-1-phosphatase.
S. Krahulec et al. Enzymes of mannitol metabolism in A. fumigatus
FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS 1265
through Fru6P reduction is not under direct control of
the cellular energy charge. Considering the results of
studies on the human pathogenic fungus Cryptococ-
cus neoformans, showing that a low Man-ol-producing
mutant strain was a 5000-fold less potent agent than the

wild type [2,3], we propose that inhibition of AfM1PDH
might be exploitable in the development of novel thera-
peutic strategies against A. fumigatus infection.
Results
Substrate specificity of Af M1PDH and Af M2DH
Purified preparations of recombinant AfM1PDH and
AfM2DH were assayed for activity in the directions of
alcohol oxidation by NAD
+
(pH 10.0) and carbonyl
group reduction by NADH (pH 7.1), with a range of
possible alternative substrates. Both enzymes showed
only trace activity for utilization of NADP
+
. Catalytic
efficiencies (in terms of k
cat
⁄ K
NADP
) were more than
three orders of magnitude below those obtained with
NAD
+
[18]. AfM1PDH and AfM2DH contain a con-
served Asp (Asp33 and Asp77, respectively) in their
coenzyme-binding pockets that is known from previous
studies of a related PSLDR, M2DH from Pseudomo-
nas fluorescens (Ps M2DH), to prevent accommodation
of the 2¢-phosphate group of NADP
+

[20,22]. Reactions
of A. fumigatus enzymes that are dependent on NADP
+
or NADPH were therefore not further investigated.
Above a level of 1% activity with Man-ol1P
(1.0 mm), AfM1PDH did not catalyze the oxidation of
Man-ol, d-sorbitol, d-ribitol, xylitol, d -xylose, l-xylose,
d-glucose, d-mannose, l-arabinose, d-arabinose,
d-galactose, l-fucose, and d-lyxose. The enzyme was
also inactive above a level of 1% activity with Fru6P
(100 mm) for reduction of Fru, l-sorbose, d-xylulose,
d-fructose 1,6-bisphosphate, d-glucose 6-phosphate
(150 mm), and d-glucose 1-phosphate (150 mm). There-
fore, AfM1PDH appears to be fairly specific for its natu-
ral pair of substrates, Man-ol1P and Fru6P. From
the highly truncated activity with Man-ol and Fru, we
conclude that the phosphate moiety in Man-ol1P and
Fru6P is essential for substrate recognition and ⁄ or
catalysis by AfM1PDH.
Alcohol oxidation by AfM2DH was assayed across
the same series of substrates utilized above with
AfM1PDH. l-Arabinitol, d-arabinitol, d-ribose, 2-deoxy-
d-galactose and 2-deoxy-d-glucose were additionally
tested as alcohol substrates. In the reduction direction,
l-sorbose, d-xylulose and dihydroxyacetone were exam-
ined. Above the 1% level of activity with Man-ol and
Fru, two polyols (d-arabinitol and d-sorbitol) and two
ketoses (d-xylulose and l-sorbose) gave significant con-
version rates. The known regioselectivity of M2DH in
the oxidation of polyols [23], indicated in Table 1,

allows assignment of d-xylulose and l-sorbose as the
products of oxidation of d-arabinitol and d-sorbitol,
respectively. Table 1 summarizes the results of kinetic
parameter determination for polyol–ketose substrate
pairs of AfM2DH. Structural comparison of polyols
that are reactive for NAD
+
-dependent oxidation by
AfM2DH with those that are not substrates of the
enzyme (Fig. S1) reveals that a d-arabo configuration is
required for a polyol to become reactive. The C2 (R)
configuration (Man-ol) is preferred over the C2 (S) con-
figuration (d-sorbitol). A model of AfM2DH was gener-
ated with the crystal structure of PsM2DH as the
template (Fig. S2) [20]. Residues contributing to the
substrate-binding site of PsM2DH are fully conserved
in AfM2DH, explaining the observed substrate specific-
ity of the A. fumigatus enzyme [12,20]. It can be assumed
from the way in which Man-ol interacts with the sub-
strate-binding site of PsM2DH in the crystal structure
that ketose substrates must bind in their open-chain
free-carbonyl form [20]. The values of K
m
and k
cat
⁄ K
m
in Table 1 are appropriately corrected for the available
proportion of reactive ketose substrate present.
Kinetic characterization

A full steady-state kinetic characterization of AfM1PDH
and AfM2DH was carried out under physiological pH
Table 1. Kinetic constants of AfM2DH for reactions with different polyol and ketose substrates. K
m
and k
cat
⁄ K
m
data for carbonyl substrates
are corrected for the available proportion of open-chain free-carbonyl forms present in aqueous solution (Fru, $ 1% [42];
D-xylulose, $ 20%
[43]; and
L-sorbose, 0.2% [44]). Numbers in parentheses show values as measured. Ketose reductions, pH 7.1; polyol oxidations, pH 10.0.
ND, not detectable (enzyme could not be saturated with
L-sorbose).
Substrate k
cat
(s
)1
) K
m
(mM) k
cat
⁄ K
m
(s
)1
ÆmM
)1
)

D-Fructose 86 ± 5 0.60 ± 0.07 (60 ± 7) 140 ± 20 (1.4 ± 0.2)
D-Xylulose 64 ± 1 1.7 ± 0.1 (8.3 ± 0.4) 39 ± 2 (7.7 ± 0.4)
L-Sorbose ND ND 1.1 ± 0.1 (0.0022 ± 0.0001)
D-Mannitol 212 ± 3 13 ± 1 17 ± 1
D-Arabinitol 162 ± 5 163 ± 13 0.99 ± 0.08
D-Sorbitol 60 ± 1 680 ± 30 0.088 ± 0.004
Enzymes of mannitol metabolism in A. fumigatus S. Krahulec et al.
1266 FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS
conditions. Lineweaver–Burk plots of initial-rate data
(Fig. 1) gave a pattern of intersecting lines, for each
enzyme and in both reaction directions, consistent with
a kinetic mechanism in which substrate and coenzyme
must bind to the enzyme to form a ternary complex
prior to the release of the first product. Kinetic param-
eters were obtained from nonlinear least-squares fits of
Eqn (3) or Eqn (4) to the experimental data. They are
summarized in Table 2, and their internal consistency
was verified with Haldane relationship analysis, com-
paring the kinetically determined reaction equilibrium
constant (K
eq
) from Eqn (7) with those reported in the
literature. Table 2 shows the useful agreement between
previously published and calculated K
eq
values [23,24].
It is interesting that, in terms of K
m
, AfM1PDH binds
Man-ol1P two orders of magnitude more tightly than

AfM2DH binds Man-ol.
Inhibition of Af M1PDH and Af M2DH by
components of the cellular energy charge
M1PDH from Escherichia coli (EcM1PDH) is evolu-
tionary related to AfM1PDH by common membership
of the family of PSLDRs [12,19]. It is strongly inhib-
ited by ATP, which acts as a competitive inhibitor
against NADH with a K
i
of $ 60 lm [25]. With a K
i
of $ 800 lm, AMP binds one order of magnitude less
strongly to EcM1PDH than does ATP [25]. To exam-
ine possible regulation of the two A. fumigatus
enzymes by components of the cellular energy charge
(ATP, ADP, and AMP), we measured inhibition of
AfM1PDH and AfM2DH by each of these adenine
nucleotides. Figure S3 shows double-reciprocal plots of
initial-rate data recorded at different concentrations of
inhibitor. The observed inhibition was, in each case,
best described by competitive binding of coenzyme and
adenine nucleotide. Inhibition constants (K
i
EI
) were
obtained from nonlinear fits of Eqn (5) to the data.
They are summarized in Table 3. Both Af M1PDH and
AfM2DH were inhibited weakly by adenine nucleo-
tides as compared with the inhibition of EcM1PDH by
ATP.

Kinetic isotope effects (KIEs)
Primary KIEs resulting from deuterium substitution of
the hydrogen atom undergoing hydride transfer from
substrate (polyol oxidation) or coenzyme (ketose
reduction) were determined for AfM1PDH and
AfM2DH at pH 7.1. Initial-rate data recorded with
protio and deuterio substrate or coenzyme were fitted
with Eqn (6), and KIEs on kinetic parameters are
AB
CD
Fig. 1. Double reciprocal plots of initial-rate data obtained for AfM1PDH (A, B) and AfM2DH (C, D) at pH 7.1 and 25 °C.
S. Krahulec et al. Enzymes of mannitol metabolism in A. fumigatus
FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS 1267
shown in Table 4. A nomenclature is used whereby
superscript D designates the KIE (e.g.
D
k
cat
). A KIE
greater than unity means that hydrogen fi deuterium
replacement caused slowing down of the enzymatic reac-
tion analyzed. NADH-dependent reduction of Fru cata-
lyzed by AfM2DH was characterized by substrate
inhibition at high Fru concentrations (K
iS
=2±1m).
Substrate inhibition was observed irrespective of
whether the NADH concentration used was saturating
or limiting in the sub-K
m

range. With the use of (4S)-
[
2
H]-NADH (NADD) instead of NADH, this substrate
inhibition disappeared (Fig. S4), precluding the use of a
single equation for calculation of the KIEs. We there-
fore obtained
D
k
cat
from a direct comparison of k
cat
data
derived from nonlinear fits of Eqn (2) and Eqn (1) with
initial rates recorded with NADH and NADD, respec-
tively and additionally by fitting Eqn (6) to the experi-
mental data obtained below the occurrence of substrate
inhibition.
D
k
cat
for Fru reduction in Table 4 (2.0 ±
0.3) represents a mean value and standard deviation for
three independent experiments evaluated in either of the
ways described above. The KIE on k
cat
⁄ K
m
for Fru was
calculated from the k

cat
and K
m
values obtained with
NADH and NADD. The KIE on k
cat
⁄ K
m
for coenzyme
was calculated as the
D
k
cat
⁄
D
K
m
ratio, where the value
of
D
K
m
was obtained by dividing K
m
data for reactions
with NADH and NADD. Note that K
m
(NADH) was
invariant (± 15%) across a wide range of Fru concen-
trations (40–800 mm), and so the choice of the constant

level of Fru was not critical for determination of
D
K
m
(1.23 ± 0.15). The KIE data in Table 4 are instrumen-
tal in delineating the kinetic mechanism of AfM1PDH
and AfM2DH, as described in the Discussion.
Free energy profile analysis for reactions
catalyzed by Af M1PDH and Af M2DH
Interpretation of the kinetic properties of an enzyme in
the context of its role in cellular metabolism relies on
knowledge of the physiological concentrations of sub-
strates, products, coenzymes and relevant effectors. We
are not aware of a study reporting intracellular metab-
olite levels in A. fumigatus (except for an early study
commenting on the cellular Man-ol content). However,
data for the closely related fungus Aspergillus niger are
available. Table 5 shows a list of metabolite concentra-
tions calculated from values in the literature, thus
defining plausible reaction conditions for AfM1PDH
and AfM2DH in vivo. Because values for the intracel-
lular concentration of Fru are not available for asper-
gilli, we approximated the level of Fru with the known
intracellular Fru concentration of Rhizobium legumin-
osarum [26]. With the assumption of the conditions in
Table 5, kinetic constants from Table 2 (including the
K
i
EI
values for ATP, ADP and AMP from Table 3)

were used to construct free energy profiles for the
transformations catalyzed by AfM1PDH and
AfM2DH. These free energy profiles (Fig. 2) show that
both enzymes operate under nonequilibrium reaction
conditions that involve a substantial thermodynamic
driving force for reduction of Fru6P and oxidation of
Table 2. Kinetic parameters of AfM1PDH and AfM2DH at 25 °C
and pH 7.1. NA, not applicable.
Parameter AfM1PDH AfM2DH
k
ox
(s
)1
) 10.6 ± 0.7 14.2 ± 0.3
K
Man-ol(1P )
(mM) 0.13 ± 0.05 11 ± 2
k
ox
⁄ K
Man-ol(1P )
(mM
)1
Æs
)1
) 80 ± 30 1.3 ± 0.2
K
NAD
(mM) 0.8 ± 0.3 0.11 ± 0.02
a

ox
1.6 ± 0.8 2.0 ± 0.4
k
red
(s
)1
) 132 ± 3 94 ± 4
K
Fru(6P )
(mM) 3.2 ± 0.2 40 ± 20
k
red
⁄ K
Fru(6P )
(mM
)1
Æs
)1
)41±3 2±1
K
NADH
(lM) 14±1 15±8
K
iNADH
(lM)2±1NA
a
red
NA 1.4 ± 0.9
1 ⁄ app K
eq

(pH 7.1)
a
300 20
K
eq
b
(M)3· 10
)10
4 · 10
)9
K
eq
c
(M)5· 10
)10
[24] 5 · 10
)9
[23]
a
The dimensionless app K
eq
at pH 7.1 was calculated with the Hal-
dane relationship (Eqn 7), using kinetic parameters from this Table.
b
The given K
eq
is pH-independent, and was obtained from the
dimensionless app K
eq
at pH 7.1.

c
Experimentally determined, pH-
independent equilibrium constant.
Table 3. Inhibition of AfM1PDH and AfM2DH by adenine nucleotides at pH 7.1 and 25 °C. K
i
EI
is a constant describing competitive inhibition
of AMP, ADP or ATP against NAD
+
or NADH. ND, not detectable.
AfM1PDH AfM2DH
Fru6P reduction Man-ol1P oxidation Fru reduction Man-ol oxidation
K
i
EI
AMP
(mM) 4.6 ± 0.6 1.5 ± 0.1 2.9 ± 0.7 5.6 ± 0.5
K
i
EI
ADP
(mM) 5.6 ± 1.9 2.0 ± 0.2 4.8 ± 1.3 5.7 ± 0.4
K
i
EI
ATP
(mM) 6.5 ± 1.4 1.4 ± 0.1 ND ND
Enzymes of mannitol metabolism in A. fumigatus S. Krahulec et al.
1268 FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS
Man-ol. Considering the uncertainty in the in vivo

levels of NADH, Fru and Man-ol1P used, we per-
formed a sensitivity analysis in which the effects of
changes in intracellular reactant concentrations on the
thermodynamic boundary conditions for the action of
AfM1PDH and AfM2DH were examined. The allowed
concentration ranges were comprehensive: 5–150 lm
NADH; 0.01–10 mm Fru; and 10–400 lm Man-ol1P.
The results (see the shaded area in Fig. 2) indicate that
the overall conclusion of this work, that the physiolog-
ical direction of the reaction of AfM1PDH is Fru6P
reduction, whereas that of AfM2DH is Man-ol oxida-
tion, was not affected by the assumed variation in the
reactant concentrations. Equations (10) and (11) are
(simplified) kinetic expressions that can be used to esti-
mate the net direction of the enzymatic reaction
(k
net
= oxidation – reduction) with the given substrate
and product concentrations. Applying the concentra-
tions from Table 5 to Eqns (10, 11), we find, for
AfM1PDH, that the direction parameter k
net
is nega-
tive or, in other words, Fru6P reduction is preferred.
With AfM2DH, by contrast, k
net
is positive, indicating
that the reaction proceeds in the direction of Man-ol
oxidation.
High-temperature stability of Af M1PDH

The literature suggests that the heat stress response of
A. fumigatus involves upregulated production of
M1PDH [9]. Because the function of AfM1PDH at ele-
vated temperatures might require pronounced resis-
tance of the enzyme to inactivation by heat, we
recorded time courses of irreversible loss of activity at
different temperatures, and use half-life times (s
H
),
calculated from these measurements (Fig. S5), as
Table 4. KIEs for reactions of AfM1PDH and AfM2DH at pH 7.1.
AfM1PDH
Man-ol1P oxidation
D
k
cat
2.9 ± 0.2
D
k
cat
⁄ K
Man-ol1P
2.4 ± 0.5
D
k
cat
⁄ K
NAD
2.4 ± 0.4
Fru6P reduction

D
k
cat
1.5 ± 0.1
D
k
cat
⁄ K
Fru6P
3.1 ± 0.4
D
k
cat
⁄ K
NADH
0.8 ± 0.2
AfM2DH
Man-ol oxidation
D
k
cat
1.0 ± 0.1
D
k
cat
⁄ K
Man-ol
1.2 ± 0.2
D
k

cat
⁄ K
NAD
1.6 ± 0.2
Fru reduction
D
k
cat
2.0 ± 0.3
D
k
cat
⁄ K
Fru
1.2 ± 0.1
D
k
cat
⁄ K
NADH
1.6 ± 0.3
Table 5. Internal metabolite concentrations from the literature.
Metabolite m
M Species Ref.
Man-ol1P 0.05
a
Aspergillus niger [30]
Fru6P 0.23 A. niger [45]
Man-ol 50
a

Aspergillus fumigatus [46]
Fru 0.4 Rhizobium leguminosarum [26]
ATP 2.6 A. niger [45]
ADP 0.47 A. niger [45]
AMP 0.09 A. niger [45]
NAD
+
0.83 A. niger [45]
NADH 0.01 A. niger [45]
a
Calculated from lmolÆg
)1
dry mycelium with application of an
intracellular volume of 1.2 mLÆg
)1
dry weight as determined for the
mycelium of A. niger [45].
AB
Fig. 2. Free energy profiles for reactions of AfM1PDH (A) and AfM2DH (B) under in vivo boundary conditions. The reaction coordinate, from
left to right, shows reduction of ketose substrate by NADH. Therefore, E, A, B, P and Q are enzyme, NADH, Fru(6P ), Man-ol(1P ) and
NAD
+
, respectively. E-A-B and E-Q-P are ternary complexes. TS is the transition state. DG values were obtained with Eqns (13)–(19), using
kinetic constants from Table 3 and 4 and reactant concentrations from Table 5 (solid line). The shaded areas between the dashed lines
depict the results of a sensitivity analysis in which the reactant levels were assumed to vary between upper and lower boundaries: NADH,
5–150 l
M; Man-ol1P, 10–400 lM; and Fru, 0.01–10 mM.
S. Krahulec et al. Enzymes of mannitol metabolism in A. fumigatus
FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS 1269
parameters for stability. The stability of AfM2DH was

analyzed in the same way, and the results for both
enzymes are summarized in Table 6. AfM1PDH is
much more stable than Af M2DH, about two orders of
magnitude in terms of s
H
at 30 °C. The stability of
AfM1PDH was hardly affected by increasing the
enzyme concentration in the assay from 0.006 mgÆmL
)1
to 0.23 mgÆmL
)1
, whereas a comparable change in
concentration for AfM2DH (0.003 mgÆmL
)1
to 0.5
mgÆmL
)1
) resulted in a substantial (approximately six-
fold) increase in s
H
. Further mechanistic analysis of
AfM1PDH and AfM2DH inactivation was beyond the
scope of this work. AfM1PDH displays remarkable sta-
bility at 40 °C and even at 50 °C, so it can be considered
to be a thermotolerant enzyme, fitting with the thermo-
tolerance of the organism. Interestingly, M1PDHs from
aspergilli (A. niger and Aspergillus parasiticus [27]) that
are less resistant to high temperature than A. fumigatus
have stabilities (at 30 °C) about one order of magnitude
below the s

H
of AfM1PDH.
Discussion
Kinetic mechanism of Af M1PDH and Af M2DH
The theory developed by Cook and Cleland is used to
deduce the kinetic mechanism of AfM1PDH and
AfM2DH from KIE data in Table 4 [28]. The pattern of
KIEs observed for AfM1PDH, in which
D
k
cat
⁄ K
mNADH
was not different from unity within the limits of error,
whereas
D
k
cat
⁄ K
mFru6P
had a large value of $ 3, indi-
cates that NADH binds to the enzyme prior to binding
of Fru6P. The absence of a KIE on k
cat
⁄ K
m
for the
substrate binding first is a clear requirement of the
strictly ordered kinetic mechanism, because, at satu-
rating concentrations of the substrate which is added

second, the commitment to catalysis becomes infinite,
and so the KIE is completely suppressed. It follows
from the additional sets of KIE data in Table 4, where
D
k
cat
⁄ K
m
values for substrate and coenzyme are both
greater than unity, that binding of Man-ol1P and
NAD
+
by AfM1PDH is not ordered, and that there is
also randomness in the binding of substrate and coen-
zyme in each direction of the reaction catalyzed by
AfM2DH. By way of comparison, an earlier study on
M1PDH from A. niger also reported random binding
of NAD
+
and Man-ol1P [29]. It is reasonable to
assume that random binding of reactants by
AfM1PDH and AfM2DH occurs in rapid equilibrium,
and the absence of curvature in Lineweaver–Burk
plots (Fig. 1) supports this notion. The parameter a
in Table 2 is therefore interpreted as a substrate–
coenzyme interaction coefficient, for which a value
greater than unity indicates that binding of one reac-
tant weakens the affinity of the enzyme for binding of
the other reactant. K
coenzyme

and K
substrate
are dissocia-
tion constants for binary enzyme complexes with coen-
zyme and substrate, respectively. K
iNADH
in Table 2 is
the dissociation constant of AfM1PDH–NADH,
whereas K
mNADH
represents an apparent binding
Table 6. Thermal stability of AfM1PDH and AfM2DH; s
H
is the half-life of the enzyme under the indicated conditions.
AfM1PDH AfM2DH
Temperature
(°C)
s
H
(h)
(0.23 mgÆmL
)1
)
a
s
H
(h)
(6 lgÆmL
)1
)

a
Temperature
(°C)
s
H
(h)
(0.5 mgÆmL
)1
)
a
s
H
(h)
(3 lgÆmL
)1
)
a
30 43 ± 4 31 ± 1 0 93 ± 7 15 ± 1
40 23 ± 1 18 ± 2 25 3.6 ± 0.2 0.64 ± 0.03
50 0.26 ± 0.03 0.16 ± 0.02 30 0.42 ± 0.04 0.06 ± 0.01
a
Protein concentration used.
Scheme 2. Steady-state reaction mechanisms for (A) AfM1PDH
and (B) AfM2DH at pH 7.1. The thick lines in (B) show the pre-
ferred paths for addition of substrate and release of product. The
dashed line shows the formation of an abortive ternary complex
(see Discussion for details).
Enzymes of mannitol metabolism in A. fumigatus S. Krahulec et al.
1270 FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS
constant. Scheme 2 summarizes the proposed kinetic

mechanisms of AfM1PDH and AfM2DH.
A value of
D
k
cat
⁄ K
m
well above unity implies that
the isotope-sensitive step of hydride transfer contrib-
utes significantly to rate limitation for the sequence of
reaction steps included in the k
cat
⁄ K
m
analyzed. For
example, k
cat
⁄ K
mFru6P
involves all steps from binding
of Fru6P to AfM1PDH–NADH up to release of the
first product, Man-ol1P or NAD
+
. In random bireac-
tant systems, k
cat
⁄ K
m
stands for reaction of the vari-
able reactant with the corresponding binary enzyme–

substrate complex. Inspection of the KIE data in
Table 4 reveals that hydride transfer is partly rate-
determining in either direction of each of the two enzy-
matic reactions. Comparison of KIEs on k
cat
and
k
cat
⁄ K
m
distinguishes datasets in which
D
k
cat
is smaller
than
D
k
cat
⁄ K
m
from others in which
D
k
cat
roughly
equals
D
k
cat

⁄ K
m
. The first case (
D
k
cat
<
D
k
cat
⁄ K
m
)
indicates that, under k
cat
conditions where the concen-
trations of coenzyme and substrate are both saturat-
ing, a reaction step not included in k
cat
⁄ K
m
,
presumably release of the second product, is partly
(
D
k
cat
> 1) or completely (
D
k

cat
= 1) rate-determining
overall. This applies to Fru6P reduction by AfM1PDH
as well as Man-ol oxidation by AfM2DH. The second
case (
D
k
cat
%
D
k
cat
⁄ K
m
> 1) indicates that hydride
transfer is rate-determining for the overall enzymatic
reaction and applies to Man-ol1P oxidation by
AfM1PDH as well as Fru reduction by AfM2DH.
Now, considering a kinetic scenario for AfM1PDH
in which k
cat
for Man-ol1P oxidation is governed by
hydride transfer, whereas k
cat
for Fru6P reduction is
partly limited by product dissociation, it is worth
remarking that the reduction k
cat
exceeds the oxidation
k

cat
by a factor of 12 (Table 2). These k
cat
conditions
imply that chemical reaction of AfM1PDH in the
reduction direction proceeds much faster than the cor-
responding chemical reaction in the oxidation direc-
tion. Under physiological pH conditions, therefore,
AfM1PDH shows a clear preference for catalysis in
the reduction direction, so the enzyme may be con-
sidered to be a Fru6P reductase. In AfM2DH, by
contrast, the turnover for Fru reduction
(k
cat
=94s
)1
) is limited by chemical transformation,
whereas the k
cat
of 14.2 s
)1
for Man-ol oxidation
reflects slow product release. With the reasonable
assumption that complete suppression of the KIE on
the oxidation k
cat
requires product release to be
minimally about 10 times slower than the hydride
transfer, chemical transformation in oxidation by
AfM2DH should occur with a rate constant of 142 s

)1
or higher. In comparison with AfM1PDH, therefore, the
kinetic properties of AfM2DH at pH 7.1 resemble much
more those expected from a true dehydrogenase acting
in the direction of NAD
+
-dependent alcohol oxidation
(see later).
Inhibition by ketose substrate during NADH-depen-
dent reduction of Fru and its absence under conditions
in which NADD is used (Fig. S4) is plausibly
explained by an expanded random kinetic mechanism
of AfM2DH (Scheme 2), involving an abortive
enzyme–NAD
+
–Fru complex, which releases NAD
+
at a rate slow enough to partially limit the overall
reaction rate. KIE data indicating that hydride transfer
is rate-determining for reduction of Fru by AfM2DH
suggest that the relative amount of enzyme–NAD
+
available for binding of Fru at the steady state can-
not be very high. However, slowing the reaction by
using NADD in place of NADH will further restrict
the availability of enzyme–NAD
+
, explaining the
complete lack of substrate inhibition under these con-
ditions.

Proposed function of Af M1PDH and Af M2DH in
mannitol metabolism
The results of free energy profile analysis strongly sup-
port the suggestion that Fru6P reduction is the pre-
ferred direction of catalytic action of AfM1PDH
in vivo, implying a physiological function of the
enzyme in Man-ol biosynthesis via path 1 of Scheme 1.
The proposed role of AfM1PDH is in good agreement
with evidence from M1PDH gene disruption studies in
other fungi, showing that Dm1pdh mutants accumulate
no Man-ol ( A. niger mycelium) [30] or have a five-fold
to 10-fold decreased Man-ol content (Alternaria alter-
nata and Phaeosphaeria nodorum) in the mycelium
[15,31]. Furthermore, A. niger undergoing sporulation
displayed 4.5-fold enhanced production of Man-ol as
compared with nonsporulating mycelium, and this
change was correlated with a similar, about six-fold,
increase in the level of M1PDH activity [30]. We also
show in this work that AfM1PDH activity is not sensi-
tive to submillimolar alterations in the levels of ATP,
ADP or AMP, indicating that, in contrast to E. coli,
where inhibition by ATP is a probable mechanism of
downregulation of M1PDH activity for Fru6P reduc-
tion [25], in A. fumigatus the cellular energy charge
exercises no control at the protein level over the rate
of Fru6P conversion into Man-ol1P. The results of
transcriptomic and proteomic analysis of the heat
shock response in A. fumigatus resulting from a shift
in growth temperature from 30 °Cto48°C suggest
that regulation of AfM1PDH activity is achieved at

the level of enzyme synthesis [9]. The marked resis-
tance of isolated AfM1PDH to inactivation by temper-
S. Krahulec et al. Enzymes of mannitol metabolism in A. fumigatus
FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS 1271
atures promoting the heat shock (40 °C and 50 °C) is
consistent with a possible role of the enzyme in confer-
ring thermotolerance to the fungus via enhanced Man-
ol production under temperature stress conditions.
Considering the reactant concentrations in Table 5,
NAD
+
-dependent oxidation of Man-ol proceeds ther-
modynamically downhill. From its kinetic properties
(Tables 1 and 2), AfM2DH appears to be well primed
for catalysis for mobilization of Man-ol (not its syn-
thesis) under these conditions. The enzyme would be
almost saturated with both alcohol substrate and
NAD
+
, whereas the assumed intracellular concentra-
tion of Fru is about two orders of magnitude below
the apparent K
m
of 40 mm. A physiological role of
AfM2DH in the utilization of intracellular Man-ol via
reversal of path 2 in Scheme 1 is therefore proposed.
In light of the suggested interplay between
AfM1PDH and AfM2DH, it is interesting that deletion
of the m1pdh gene in some fungi resulted in poor
growth (A. alternata) [31] or the absence thereof

(P. nodorum) [15] on Man-ol as carbon source. The
corresponding Dm2dh mutants, however, showed sub-
stantial growth under these conditions [15,31]. It is
possible, therefore, that M1PDH has an additional
physiological function in the catabolism of external
Man-ol once the substrate has become phosphorylated
during or after uptake into the cell. However, a clear
requirement for AfM1PDH to change directional pref-
erence in catalytic action would be that levels of intra-
cellular metabolites undergo a large shift from the
situation portrayed in Table 5 to another one that
favors Man-ol1P, NAD
+
or both.
Are oxidoreductases other than AfM1PDH and
AfM2DH involved in the metabolism of mannitol by
A. fumigatus? We searched the genome of the organ-
ism, and identified an ORF whose translated product
is 35.2% identical in amino acid sequence to the
M2DH from Agaricus bisporus (AbM2DH) [12]. This
putative protein of A. fumigatus is currently annotated
as Sou1-like sorbitol ⁄ xylulose reductase (UniProt⁄
TrEMBL entry Q4WZX5). In contrast to AfM2DH,
AbM2DH is an NADP
+
-dependent enzyme. From its
sequence and three-dimensional structure, AbM2DH is
classified as member of the short-chain dehydroge-
nase ⁄ reductase superfamily of proteins and enzymes,
and shows no significant evolutionary relationship with

AfM2DH. Now, the tentative existence of an
NADP
+
-dependent M2DH in A. fumigatus made it
necessary to examine the possibility that Man-ol is
produced from Fru by reduction with NADPH. How-
ever, assuming a value of 0.62 for the ratio of intracel-
lular concentrations of NADPH and NADP
+
(data
from A. niger [32]) and applying the values for the
in vivo levels of Man-ol and Fru from Table 5, we
reach the immediate conclusion that reduction of Fru
by NADPH is not thermodynamically feasible under
these conditions. In other words, AfM1PDH is the key
enzyme for Man-ol biosynthesis in A. fumigatus,
irrespective of a possible multiplicity of NADP
+
⁄
NAD
+
-dependent M2DH activities in the organism. A
side remark in this respect is that gene expression and
gene disruption studies in P. nodorum and A. niger
could possibly have overlooked the existence of the
NAD
+
-dependent M2DH ( P. nodorum, Q0UEB6;
A. niger, A2QGA1) [15,33].
Features of Af M1PDH structure and function that

might be exploited for inhibition
Inhibition of the fungal biosynthesis of Man-ol is a
promising strategy for development of new therapies
against infection by A. fumigatus. The results of this
work show that antagonism of AfM1PDH presents a
clear target for achieving the desired inhibition.
Moreover, because the human genome does not
encode proteins homologous to AfM1PDH or any
other protein of the current PSLDR family, there is
a good chance that compounds raised against
AfM1PDH will not show substantial cross-reactivity
with respect to binding of proteins of the human
host. The high specificity of AfM1PDH for reactions
with phosphorylated substrates could be a feature of
recognition to be exploited in the design of selective
substrate and ⁄ or transition state analogs. The N-ter-
minal NAD
+
-binding domain of AfM1PDH could be
another target for selective inhibition, given that, in
PSLDRs, this domain adopts a somewhat unusual
Rossmann fold that is distinct in many details from
isofunctional domains in enzymes of other dehydroge-
nase ⁄ reductase families. Precedents from the litera-
ture, on lactate dehydrogenase [34] for example, are
highly encouraging in showing that, by using inhibi-
tors targeted towards the coenzyme-binding site, it
may be possible to achieve inhibition that is not only
selective for a particular enzyme type, as would be
necessary for inhibition of AfM1PDH, but can even

discriminate between the same enzyme from parasite
and host.
Experimental procedures
Materials
Recombinant AfM1PDH and AfM2DH were produced in
E. coli and purified as described recently [17,18]. Unless
otherwise indicated, highly purified preparations of recom-
Enzymes of mannitol metabolism in A. fumigatus S. Krahulec et al.
1272 FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS
binant AfM1PDH and AfM2DH were used in all
experiments. Enzymatic production of Man-ol1P and
5-[
2
H]-Man-ol1P was carried out with previously reported
methods [17]. d-Xylulose was synthesized by microbial
oxidation of d-arabinitol, employing a previously described
protocol [35]. NADD was prepared with an enzymatic
procedure that used glucose dehydrogenase from Baci-
llus megaterium and 1-[
2
H]-d-glucose. Deutero-NADH was
purified by MonoQ anion exchange chromatography, as
previously described [23,36,37]. The optical purity of
NADD and its degree of deuteration (95 ± 1%) were ana-
lyzed by
1
H-NMR and MS, respectively. 2-[
2
H]-Man-ol was
prepared by enzymatic conversion of Fru, and was purified

as described recently [38,39]. The isotopic purity of 2-[
2
H]-
Man-ol was determined by MS (> 99%). No residual Fru,
NAD
+
or NADH was detected by
13
C-NMR. Man-ol,
Fru, Fru6P, b-nicotinamide adenine dinucleotides (NAD
+
and NADH) and adenine nucleotides (ATP, ADP and
AMP) at a purity ‡ 95% were obtained from commercial
sources.
Assays
Protein concentrations were determined with the Bio-Rad
protein assay (Bio-Rad Laboratories, Hercules, CA, USA)
referenced against known concentrations of BSA. Initial-
rate data were collected with a DU800 spectrophotometer
(Beckman Coulter, Fullerton, CA, USA) at 25 °C, and are
based on the measurement of formation or depletion of
NAD(P)H at 340 nm (e
NADH
= 6.22 cm
)1
Æmm
)1
). Unless
otherwise indicated, substrate screening was performed at a
constant concentration of 300 mm. Tris ⁄ HCl buffer

(100 mm; pH 7.1) was used for ketose reduction. Gly-
cine ⁄ NaOH buffer (100 mm; pH 10.0) was used for polyol
oxidation. Different pH conditions were chosen because
alcohol oxidation by NAD
+
generally proceeds best at high
pH, whereas a lower pH value is normally suitable for car-
bonyl group reduction by NADH. The concentrations of
NADH and NAD
+
used in these assays were 0.2 and
2.0 mm, respectively. Apparent kinetic parameters (k
cat
and
K
m
) were obtained for those compounds that had shown
significant conversion rates in screening assays, using a
threshold of 1% of the activity with the respective native
substrate (Man-ol and Fru; Man-ol1P and Fru6P). By
application of this criterion, the following substrates were
selected for reaction with AfM2DH in the given concentra-
tion range: Fru (2–1000 mm), d-xylulose (0.5–450 mm), l-
sorbose (20–1000 mm), Man-ol (1–400 mm), d-arabinitol
(3–1300 mm) and d-sorbitol (30–1600 mm).
Tris ⁄ HCl buffer (100 mm; pH 7.1) was used in all further
kinetic studies. Full kinetic characterization of AfM1PDH
involved initial-rate measurements under conditions of vari-
ous concentrations of Fru6P (0.17–8.7 mm) and Man-ol1P
(0.02–2.3 mm) at several constant concentrations of NADH

(0.0017–0.17 mm) and NAD
+
(0.06–5.9 mm), respectively.
Measurements performed with AfM2DH involved various
concentrations of Fru (8.5–430 mm) and Man-ol (3–110 mm)
at several constant concentrations of NADH (0.014–0.20
mm) and NAD
+
(0.085–1.4 mm), respectively. Inhibition by
adenine nucleotides (AMP, ADP and ATP) was analyzed by
measuring initial rates under conditions in which the concen-
tration of NADH (0.008–0.25 mm) or NAD
+
(0.05–2.5 mm)
was varied at several constant concentrations of the
respective adenine nucleotide in the range 0.63–5.0 mm. The
concentration of carbonyl or polyol substrates was constant
and saturating.
Primary deuterium KIEs on apparent kinetic parameters
of AfM1PDH and AfM2DH were obtained from a compari-
son of initial rates recorded with unlabeled or deuterium-
labeled substrates or coenzymes. Oxidation of Man-ol1P
and 5-[
2
H]-Man-ol1P was measured under conditions in
which the concentration of NAD
+
(0.08–8 mm) or Man-
ol1P ⁄ 5-[
2

H]-Man-ol1P (0.04–6.2 mm) was varied at a con-
stant and saturating concentration of the respective other
substrate (NAD
+
, 5.7 mm; Man-ol1P ⁄ 5-[
2
H]-Man-ol1P,
1.0 mm). Fru6P reduction was measured under conditions in
which the concentration of NADH ⁄ NADD (0.012–0.2 mm)
or Fru6P (0.45–45 mm) was varied at a constant and saturat-
ing concentration of the respective other substrate (NADH ⁄
NADD, 0.2 mm; Fru6P,45mm). Likewise, the conditions
used for determination of KIEs on kinetic parameters for
AfM2DH were as follows. Oxidation: Man-ol ⁄ 2-[
2
H]-Man-ol,
0.9–180 mm, and NAD
+
,4mm; NAD
+
, 0.08–4 mm, and
Man-ol ⁄ 2-[
2
H]-Man-ol, 260 mm. Reduction: Fru, 4–840 mm,
and NADH ⁄ NADD, 0.25 mm; NADH ⁄ NADD, 0.002–0.2
mm, and Fru, 800 mm.
Data processing
Kinetic parameters were obtained from a nonlinear fit of
the appropriate equation to the data. Unweighted nonlinear
least-squares regression analysis with sigma plot 9.0

(SYSTAT Software; San Jose, CA, USA) was used. In
Eqns (1)-(6), v is the initial rate, k
cat
is the kinetic turnover
number, E and S are the molar concentrations of enzyme
and substrate, K
m
is an apparent Michaelis constant, and
K
iS
is a substrate inhibition constant. E was obtained from
the protein concentration, using molecular masses of
44.2 kDa and 57.6 kDa for AfM1PDH and AfM2DH,
respectively [17,18]. Equation (3) implies ordered binding of
substrates A and B in a bisubstrate reaction where K
iA
is
the dissociation constant for A. Equation (4) is used for
random bisubstrate kinetics, where K
A
and K
B
are dissocia-
tion constants for A and B, and a is a factor describing
how bound A affects the binding of B. In Eqn (5), K
i
EI
is a
competitive inhibition constant, and I is the molar inhibitor
concentration. Unless mentioned, KIEs were obtained by

using Eqn (6) [40], where E
V
and E
V ⁄ K
are isotope effects
minus 1 on k
cat
and k
cat
⁄ K
m
, respectively. F
i
is the fraction
of deuterium in the labeled substrate.
S. Krahulec et al. Enzymes of mannitol metabolism in A. fumigatus
FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS 1273
v ¼ k
cat
Á E Á S= ðK
m
þ SÞð1Þ
v ¼ k
cat
Á E Á S=½K
m
þ S Áð1 þ S=K
iS
Þ ð2Þ
v ¼ k

cat
Á E Á A Á B=ðK
iA
Á K
mB
þ K
mA
Á B þ K
mB
Á A þ A Á BÞ
ð3Þ
v ¼ k
cat
Á E Á A Á B=ðA Á B þ a Á K
A
Á Bþa Á K
B
Á A þ a Á K
A
Á K
B
Þ
ð4Þ
v ¼ k
cat
Á E Á S=½K
m
Áð1 þ I=K
EI
i

ÞþSð5Þ
v ¼ k
cat
Á E Á S=½K
m
Áð1 þ F
i
Á E
V=K
ÞþS Áð1 þ F
i
Á E
V
Þ ð6Þ
Equation (7) is the Haldane relationship for an ordered
bi-bi kinetic mechanism, where app K
eq
is the (kinetically
determined) equilibrium constant of the reaction; k
ox
and
k
red
are k
cat
values for alcohol oxidation and ketose reduc-
tion; K
iNADH
and K
iNAD

are dissociation constants for
NADH and NAD
+
; and K
mRO
and K
mROH
are Michaelis
constants for ketose and polyol. Note: in the random bi-
substrate mechanism, the expression aÆK
A
ÆK
B
is used to
replace K
iA
ÆK
mB
in Eqn (7). The combined effect of com-
petitive inhibition by adenine nucleotides is accounted for
by Eqn (8) or Eqn (9), where an asterisk indicates the
apparent binding constant. Equations (10) and (11) are
simplified expressions of enzyme directional preference
(k
net
), based solely on catalytic efficiencies. Formally, they
are derived from the complete rate equation for a rapid
equilibrium random bireactant kinetic mechanism [41], by
leaving out all terms in the denominator. Strictly, these
equations would be applicable only for limiting reactant

concentrations. However, they are qualitatively useful
because, at given reactant concentrations, a positive value
of k
net
indicates a preference for alcohol oxidation,
whereas a negative value signifies a preference for ketose
reduction. Note that the term aÆK
A
ÆK
B
applies to a ran-
dom mechanism, as in Eqn (10). When the mechanism is
ordered, the aÆK
A
ÆK
B
term is replaced by K
iA
ÆK
mB
,asin
Eqn (11). Gibbs free energy (DG,kJÆmol
)1
) profiles for
reactions catalyzed by AfM2DH and AfM1PDH at
pH 7.1 were constructed with the use of Eqns (12)-(19). R
is the gas constant (0.008314 kJÆmol
)1
ÆK
)1

), and T is the
temperature (298.15 K). The parameter K in Eqn (12) is
the mass action ratio calculated by applying intracellular
concentrations of reactants. DG
Keff
is the difference
between DG at K
eq
and DG at a given value of K. Calcu-
lation of DG values at ternary complexes (indicated by
subscript) and the transition state (subscript TS) is shown
in Eqns (16)–(19). k and h are the Boltzmann constant
(1.38 · 10
)26
kJÆK
)1
) and the Planck constant
(6.63 · 10
)37
kJÆs), respectively.
appK
eq
¼ k
ox
Á K
iNADH
Á K
mRO
=ðk
red

Á K
iNAD
Á K
mROH
Þð7Þ
K
NADðHÞ
Ã
¼ K
NADðHÞ
Áð1 þ AMP=K
EI
i AMP
þ ADP=K
EI
i ADP
þ ATP=K
EI
i ATP
Þ
ð8Þ
K
iNADH
Ã
¼ K
iNADH
Áð1 þ AMP=K
EI
i AMP
þ ADP=K

EI
i ADP
þ ATP=K
EI
i ATP
Þ
ð9Þ
k
net M2DH
¼ k
ox
Á NAD
þ
Á Man- ol=ða
ox
Á K
NAD
Ã
Á K
Man-ol
ÞÀk
red
Á NADH Á Fru=
ða
red
Á K
NADH
Ã
Á K
Fru

Þ
ð10Þ
k
net M1PDH
¼ k
ox
Á NAD
þ
Á Man-ol1P=
ða
ox
Á K
NAD
Ã
Á K
Man-ol1P
Þ
À k
red
Á NADH Á Fru6P=ðK
iNADH
Ã
Á K
mFru6P
Þ
ð11Þ
K ¼ NAD
þ
Á ROH=ðNADH Á RO Þð12Þ
DG

K
eq
¼ÀR Á T Á lnð1=K
eq
Þð13Þ
DG
K
¼ÀR Á T Á lnðKÞð14Þ
DG
Keff
¼ DG
Keq
ÀDG
K
ð15Þ
DG
E-NAD-ROH
¼ R Á T Á ln½K
NAD
Ã
Á K
ROH
Á a=
ðNAD
þ
Á ROHÞ þ DG
Keff
ð16Þ
DG
E-NADH-RO

¼ R Á T Á ln½K
iNADH
Ã
Á K
mRO
=ðNADH Á ROÞ
ð17Þ
DG
E-NADH-RO
¼ R Á T Á ln½K
NADH
Ã
Á K
RO
Á a= ðNADH Á ROÞ
ð18Þ
DG
TS
¼ÀR Á T Á ln½k
ox
Á NAD
þ
Á ROH=ðK
NAD
Á K
ROH
Á aÞ
þ R Á T Á lnðk Á T=hÞþDG
Keff
ð19Þ

Acknowledgements
V. Pacher and K. Longus are thanked for expert tech-
nical assistance. We are grateful to M. Murkovic
(Institute of Biochemistry, Graz University of Technol-
ogy) and H. Weber (Institute of Organic Chemistry,
Graz University of Technology) for MS and NMR
measurements, respectively. Financial support from the
Austrian Science Fund FWF (P18275-B09 to B. Nide-
tzky) is gratefully acknowledged.
References
1 Solomon PS, Waters OD & Oliver RP (2007) Decoding
the mannitol enigma in filamentous fungi. Trends
Microbiol 15 , 257–262.
Enzymes of mannitol metabolism in A. fumigatus S. Krahulec et al.
1274 FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS
2 Chaturvedi V, Flynn T, Niehaus WG & Wong B (1996)
Stress tolerance and pathogenic potential of a mannitol
mutant of Cryptococcus neoformans. Microbiology
142(Pt 4), 937–943.
3 Chaturvedi V, Wong B & Newman SL (1996) Oxidative
killing of Cryptococcus neoformans by human neutro-
phils. Evidence that fungal mannitol protects by
scavenging reactive oxygen intermediates. J Immunol
156, 3836–3840.
4Ve
´
l
€
ez H, Glassbrook NJ & Daub ME (2008) Mannitol
biosynthesis is required for plant pathogenicity by

Alternaria alternata. FEMS Microbiol Lett 285,
122–129.
5 Jennings DB, Ehrenshaft M, Pharr DM & Williamson
JD (1998) Roles for mannitol and mannitol dehydroge-
nase in active oxygen-mediated plant defense. Proc Natl
Acad Sci USA 95, 15129–15133.
6 Voegele RT, Hahn M, Lohaus G, Link T, Heiser I &
Mendgen K (2005) Possible roles for mannitol and
mannitol dehydrogenase in the biotrophic plant patho-
gen Uromyces fabae. Plant Physiol 137, 190–198.
7 Latge
´
JP (1999) Aspergillus fumigatus and aspergillosis.
Clin Microbiol Rev 12, 310–350.
8 Wong B, Brauer KL, Tsai RR & Jayasimhulu K (1989)
Increased amounts of the Aspergillus metabolite d-man-
nitol in tissue and serum of rats with experimental
aspergillosis. J Infect Dis 160, 95–103.
9 Albrecht D, Guthke R, Brakhage AA & Kniemeyer O
(2010) Integrative analysis of the heat shock response
in Aspergillus fumigatus. BMC Genomics 11: 32,
doi:10.1186/1471-2164-11-32.
10 Stevens DA, Moss RB, Kurup VP, Knutsen AP, Green-
berger P, Judson MA, Denning DW, Crameri R, Brody
AS, Light M et al. (2003) Allergic bronchopulmonary
aspergillosis in cystic fibrosis – state of the art: Cystic
Fibrosis Foundation Consensus Conference. Clin Infect
Dis 37(Suppl. 3), 225–264.
11 Shen B, Jensen RG & Bohnert HJ (1997) Mannitol pro-
tects against oxidation by hydroxyl radicals. Plant Phys-

iol 115, 527–532.
12 Nidetzky B & Klimacek M (2007) Fungal mannitol 2-de-
hydrogenases and mannitol-1-phosphate 5-dehydro-
genases constitute novel branches in the protein family of
polyol-specific long-chain dehydrogenases and reductases.
In Enzymology and Molecular Biology of Carbonyl Meta-
bolism (Weiner H, Maser E, Lindahl R & Plapp B eds), pp
305–322. Purdue University Press, West Lafayette.
13 Ueng ST, Hartanowicz P, Lewandoski C, Keller J,
Holick M & McGuinness ET (1976) d-Mannitol
dehydrogenase from Absidia glauca. Purification,
metabolic role, and subunit interactions. Biochemistry
15, 1743–1749.
14 Lee WH (1967) Mannitol acetyl phosphate phospho-
transferase of Aspergillus
. Biochem Biophys Res Com-
mun 29, 337–342.
15 Solomon PS, Waters OD, Jorgens CI, Lowe RG,
Rechberger J, Trengove RD & Oliver RP (2006)
Mannitol is required for asexual sporulation in the
wheat pathogen Stagonospora nodorum (glume blotch).
Biochem J 399, 231–239.
16 Strandberg GW (1969) d-mannitol metabolism by
Aspergillus candidus. J Bacteriol 97, 1305–1309.
17 Krahulec S, Armao GC, Weber H, Klimacek M &
Nidetzky B (2008) Characterization of recombinant
Aspergillus fumigatus mannitol-1-phosphate 5-dehydro-
genase and its application for the stereoselective synthe-
sis of protio and deuterio forms of d-mannitol 1-
phosphate. Carbohydr Res 343, 1414–1423.

18 Krahulec S, Armao GC, Bubner P, Klimacek M &
Nidetzky B (2009) Polyol-specific long-chain
dehydrogenases ⁄ reductases of mannitol metabolism in
Aspergillus fumigatus: biochemical characterization and
pH studies of mannitol 2-dehydrogenase and mannitol-
1-phosphate 5-dehydrogenase. Chem Biol Interact 178,
274–282.
19 Klimacek M, Kavanagh KL, Wilson DK & Nidetzky B
(2003) Pseudomonas fluorescens mannitol 2-dehydroge-
nase and the family of polyol-specific long-chain dehy-
drogenases ⁄ reductases: sequence-based classification and
analysis of structure–function relationships. Chem Biol
Interact 143–144, 559–582.
20 Kavanagh KL, Klimacek M, Nidetzky B & Wilson DK
(2002) Crystal structure of Pseudomonas fluorescens
mannitol 2-dehydrogenase binary and ternary com-
plexes. Specificity and catalytic mechanism. J Biol Chem
277, 43433–43442.
21 Kavanagh KL, Klimacek M, Nidetzky B & Wilson DK
(2003) Crystal structure of Pseudomonas fluorescens
mannitol 2-dehydrogenase: evidence for a very divergent
long-chain dehydrogenase family. Chem Biol Interact
143–144, 551–558.
22 Bubner P, Klimacek M & Nidetzky B (2008) Structure-
guided engineering of the coenzyme specificity of Pseu-
domonas fluorescens mannitol 2-dehydrogenase to enable
efficient utilization of NAD(H) and NADP(H). FEBS
Lett 582, 233–237.
23 Slatner M, Nidetzky B & Kulbe KD (1999) Kinetic
study of the catalytic mechanism of mannitol dehydro-

genase from Pseudomonas fluorescens. Biochemistry 38,
10489–10498.
24 Wolff JB & Kaplan NO (1956) d-Mannitol 1-phosphate
dehydrogenase from Escherichia coli. J Biol Chem 218,
849–869.
25 Klungsøyr L (1967) The binding of adenine nucleotides
to the mannitol-I-phosphate dehydrogenase of Escheri-
chia coli. Biochim Biophys Acta 146, 10–17.
26 Glenn AR, Arwas R, McKay IA & Dilworth MJ (1984)
Fructose metabolism in wild-type, fructokinase-negative
and revertant strains of Rhizobium leguminosarum.
J Gen Microbiol 130, 231–237.
S. Krahulec et al. Enzymes of mannitol metabolism in A. fumigatus
FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS 1275
27 Foreman JE and Niehaus WG Jr (1985) Zn
2+
-induced
cooperativity of mannitol-1-phosphate dehydrogenase
from Aspergillus parasiticus. J Biol Chem 260, 10019–
10022.
28 Cook PF (1991) Kinetic and regulatory mechanisms of
enzymes from isotope effects. In Enzyme Mechanism
from Isotope Effects (Cook PF ed), pp 203–231. CRC
Press, Boca Raton.
29 Kiser RC & Niehaus WG Jr (1981) Purification and
kinetic characterization of mannitol-1-phosphate dehy-
drogenase from Aspergillus niger. Arch Biochem Biophys
211, 613–621.
30 Ruijter GJG, Bax M, Patel H, Flitter SJ, van de
Vondervoort PJI, de Vries RP, vanKuyk PA & Visser J

(2003) Mannitol is required for stress tolerance in Asper-
gillus niger conidiospores. Eukaryot Cell 2, 690–698.
31 Ve
´
l
€
ez H, Glassbrook NJ & Daub ME (2007) Mannitol
metabolism in the phytopathogenic fungus Alternaria
alternata. Fungal Genet Biol 44, 258–268.
32 Poulsen BR, Nohr J, Douthwaite S, Hansen LV,
Iversen JJL, Visser J & Ruijter GJG (2005) Increased
NADPH concentration obtained by metabolic engineer-
ing of the pentose phosphate pathway in Aspergillus
niger. FEBS J 272, 1313–1325.
33 Aguilar-Osorio G, Vankuyk PA, Seiboth B, Blom D,
Solomon PS, Vinck A, Kindt F, Wo
¨
sten HA & de Vries
RP (2010) Spatial and developmental differentiation of
mannitol dehydrogenase and mannitol-1-phosphate
dehydrogenase in Aspergillus niger. Eukaryot Cell 9,
1398–1402.
34 Vander Jagt DL, Deck LM & Royer RE (2000) Gossy-
pol: prototype of inhibitors targeted to dinucleotide
folds. Curr Med Chem 7, 479–498.
35 Mayer G, Kulbe KD & Nidetzky B (2002) Utilization
of xylitol dehydrogenase in a combined microbial ⁄ enzy-
matic process for production of xylitol from d-glucose.
Appl Biochem Biotechnol 98–100, 577–589.
36 Mostad SB & Glasfeld A (1993) Using high field NMR

to determine dehydrogenase stereospecificity with
respect to NADH. J Chem Educ 70, 504–506.
37 Orr GA & Blanchard JS (1984) High-performance
ion-exchange separation of oxidized and reduced
nicotinamide adenine dinucleotides. Anal Biochem 142,
232–234.
38 Klimacek M & Nidetzky B (2002) Examining the rela-
tive timing of hydrogen abstraction steps during
NAD
+
-dependent oxidation of secondary alcohols cata-
lyzed by long-chain d-mannitol dehydrogenase from
Pseudomonas fluorescens using pH and kinetic isotope
effects. Biochemistry 41, 10158–10165.
39 Klimacek M & Nidetzky B (2010) The oxyanion hole
of Pseudomonas fluorescens mannitol 2-dehydrogenase:
a novel structural motif for electrostatic stabilization in
alcohol dehydrogenase active sites. Biochem J 425, 455–
463.
40 Blanchard JS & Cleland WW (1980) Kinetic and
chemical mechanisms of yeast formate dehydrogenase.
Biochemistry 19, 3543–3550.
41 Segel IH (1993) Enzyme Kinetics – Behavior and
Analysis of Rapid Equilibrium and Steady-State Enzyme
Systems. Wiley, New York.
42 Collins P & Ferrier R (1995) Monosaccharides: Their
Chemistry and Their Roles in Natural Products. Wiley,
Chichester.
43 Wu J, Serianni AS & Vuorinen T (1990) Furanose ring
anomerization: kinetic and thermodynamic studies of

the d-2-pentuloses by 13C-n.m.r. spectroscopy.
Carbohydr Res 206, 1–12.
44 Hayward LD & Angyal SJ (1977) A symmetry rule for
the circular dichroism of reducing sugars, and the pro-
portion of carbonyl forms in aqueous solutions thereof.
Carbohydr Res 53, 13–20.
45 Ruijter GJG & Visser J (1996) Determination of inter-
mediary metabolites in Aspergillus niger. J Microbiol
Methods 25, 295–302.
46 Yamamoto Y, Nitta K, Tango K, Saito T & Tsuchim-
uro M (1965) Studies on the metabolic products of a
strain of Aspergillus fumigatus (DH 413). I. Isolation
and chemical structures of metabolites. Chem Pharm
Bull (Tokyo) 13, 935–941.
Supporting information
The following supplementary material is available:
Fig. S1. Structural comparison of polyol substrates of
AfM2DH.
Fig. S2. Comparison of the structural model of
AfM2DH with the experimentally determined crystal
structure of PsM2DH (1lj8).
Fig. S3. Kinetic analysis of inhibition of AfM1PDH
(A–F) and AfM2DH (G–L) by adenine nucleotides at
pH 7.1.
Fig. S4. Michaelis–Menten plot for Fru reduction by
AfM2DH employing NADH or NADD.
Fig. S5. Irreversible inactivation of AfM1PDH (A, B)
and AfM2DH (C, D) at different temperatures, repre-
sented as semilogarithmic plots.
This supplementary material can be found in the

online version of this article.
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should be addressed to the authors.
Enzymes of mannitol metabolism in A. fumigatus S. Krahulec et al.
1276 FEBS Journal 278 (2011) 1264–1276 ª 2011 The Authors Journal compilation ª 2011 FEBS