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

Primary kinetic isotope effects resulting from deuteration of alco-hol substrate or NADH showed that, for AfM1PDH, binding ofD -manni-tol 1-phosphate and NAD+ is random, whereas D-fructo

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: bernd.nidetzky@TUGraz.at

(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 25C and

pH 7.1 Primary kinetic isotope effects resulting from deuteration of alco-hol substrate or NADH showed that, for AfM1PDH, binding ofD -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 (kcat= 10.6 s)1) as well asD-fructose reduction by AfM2DH (kcat= 94 s)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 AfM2DH 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 40C 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)-[2H]-NADH; PSLDR, polyol-specific long-chain dehydrogenase ⁄ reductase; PsM2DH, mannitol 2-dehydrogenase from Pseudomonas fluorescens.

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.

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 kcat⁄ KNADP) 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 (PsM2DH), 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 Kmand kcat⁄ Km

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).

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 (Keq) from Eqn (7) with those reported in the

literature Table 2 shows the useful agreement between

previously published and calculated Keqvalues [23,24]

It is interesting that, in terms of Km, 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 Ki of  60 lm [25] With a Ki

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 (KiEI) were obtained from nonlinear fits of Eqn (5) to the data They are summarized in Table 3 Both AfM1PDH 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

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.

shown in Table 4 A nomenclature is used whereby

superscript D designates the KIE (e.g Dkcat) A KIE

greater than unity means that hydrogen fi deuterium

replacement caused slowing down of the enzymatic

reac-tion analyzed NADH-dependent reducreac-tion of Fru

cata-lyzed by AfM2DH was characterized by substrate

inhibition at high Fru concentrations (KiS= 2 ± 1 m)

Substrate inhibition was observed irrespective of

whether the NADH concentration used was saturating

or limiting in the sub-Km range With the use of

(4S)-[2H]-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 obtainedDkcatfrom a direct comparison of kcatdata

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 Dkcat 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 kcat⁄ Kmfor Fru was calculated from the kcat and Km values obtained with NADH and NADD The KIE on kcat⁄ Kmfor coenzyme was calculated as theDkcat⁄DKmratio, where the value

ofDKmwas obtained by dividing Kmdata for reactions with NADH and NADD Note that Km(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 DKm (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

KiEI 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.

K Man-ol(1P ) (m M ) 0.13 ± 0.05 11 ± 2

kox⁄ K Man-ol(1P ) (m M )1Æs)1) 80 ± 30 1.3 ± 0.2

KFru(6P )(m M ) 3.2 ± 0.2 40 ± 20

k red ⁄ K Fru(6P ) (m M )1Æs)1) 41 ± 3 2 ± 1

KiNADH(l M ) 2 ± 1 NA

Keqc ( M ) 5 · 10)10[24] 5 · 10)9[23]

a The dimensionless app Keqat 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 Keqat 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 iEIis a constant describing competitive inhibition

of AMP, ADP or ATP against NAD+or NADH ND, not detectable.

KiEI

KiEI

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 (knet= 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 knet is nega-tive or, in other words, Fru6P reduction is preferred With AfM2DH, by contrast, knetis 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 (sH), 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 kcat⁄ K Man-ol1P 2.4 ± 0.5

D

Fru6P reduction

D

k cat ⁄ K Fru6P 3.1 ± 0.4

AfM2DH

Man-ol oxidation

D

D kcat⁄ K Man-ol 1.2 ± 0.2

Fru reduction

D

Table 5 Internal metabolite concentrations from the literature.

a

Calculated from lmolÆg)1 dry mycelium with application of an

intracellular volume of 1.2 mLÆg)1dry weight as determined for the

mycelium of A niger [45].

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 l M ; and Fru, 0.01–10 m M

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 AfM2DH, about two orders of

magnitude in terms of sH at 30C 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 sH Further mechanistic analysis of

AfM1PDH and AfM2DH inactivation was beyond the

scope of this work AfM1PDH displays remarkable

sta-bility at 40C 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 30C) about one order of magnitude

below the sHof 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 whichDkcat⁄ KmNADH

was not different from unity within the limits of error,

whereas Dkcat⁄ KmFru6P 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 kcat⁄ Km 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

Dkcat⁄ Km 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 Kcoenzyme and Ksubstrate are dissocia-tion constants for binary enzyme complexes with coen-zyme and substrate, respectively KiNADH in Table 2 is the dissociation constant of AfM1PDH–NADH, whereas KmNADH 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.

Temperature

(C)

sH(h) (0.23 mgÆmL)1) a

sH(h) (6 lgÆmL)1) a

Temperature (C)

sH(h) (0.5 mgÆmL)1) a

sH(h) (3 lgÆmL)1) a

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).

constant Scheme 2 summarizes the proposed kinetic

mechanisms of AfM1PDH and AfM2DH

A value of Dkcat⁄ Km 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 kcat⁄ Km analyzed For

example, kcat⁄ KmFru6P 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, kcat⁄ Km 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 kcat and

kcat⁄ Kmdistinguishes datasets in whichDkcatis smaller

than Dkcat⁄ Km from others in which Dkcat roughly

equals Dkcat⁄ Km The first case (Dkcat<Dkcat⁄ Km)

indicates that, under kcatconditions where the

concen-trations of coenzyme and substrate are both

saturat-ing, a reaction step not included in kcat⁄ Km,

presumably release of the second product, is partly

(Dkcat> 1) or completely (Dkcat= 1) rate-determining

overall This applies to Fru6P reduction by AfM1PDH

as well as Man-ol oxidation by AfM2DH The second

case (DkcatDkcat⁄ Km> 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 kcat for Man-ol1P oxidation is governed by

hydride transfer, whereas kcat for Fru6P reduction is

partly limited by product dissociation, it is worth

remarking that the reduction kcatexceeds the oxidation

kcat by a factor of 12 (Table 2) These kcat 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

(kcat= 94 s)1) is limited by chemical transformation,

whereas the kcat 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 kcat 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 30C to 48 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-atures promoting the heat shock (40C 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 Km 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-binant AfM1PDH and AfM2DH were used in all

experiments Enzymatic production of Man-ol1P and

5-[2H]-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-[2H]-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 by1H-NMR and MS, respectively 2-[2H]-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 13C-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 25C, and are

based on the measurement of formation or depletion of

NAD(P)H at 340 nm (eNADH= 6.22 cm)1Æmm)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 (kcatand

Km) 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-[2H]-Man-ol1P was measured under conditions in which the concentration of NAD+ (0.08–8 mm) or Man-ol1P⁄ 5-[2H]-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-[2H]-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, 45 mm) 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+, 4 mm; NAD+, 0.08–4 mm, and Man-ol⁄ 2-[2H]-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 plot9.0 (SYSTAT Software; San Jose, CA, USA) was used In Eqns (1)-(6), v is the initial rate, kcat is the kinetic turnover number, E and S are the molar concentrations of enzyme and substrate, Km is an apparent Michaelis constant, and

KiSis 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 KiA is the dissociation constant for A Equation (4) is used for random bisubstrate kinetics, where KAand KBare dissocia-tion constants for A and B, and a is a factor describing how bound A affects the binding of B In Eqn (5), KiEIis a competitive inhibition constant, and I is the molar inhibitor concentration Unless mentioned, KIEs were obtained by using Eqn (6) [40], where EV and EV ⁄ Kare isotope effects minus 1 on kcatand kcat⁄ Km, respectively Fiis the fraction

of deuterium in the labeled substrate