Multiple Forms of Xylose Reductase in Candida intermedia: Comparison of Their Functional Properties Using Quantitative Structure-Activity Relationships, Steady-State Kinetic Analysis, and pH Studies

Article abstract of DOI:10.1021/jf034426j

The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer NADH ≈4-fold over NADPH (dual specific xylose reductase; dsXR). To compare the functional properties of the isozymes, a steady-state kinetic analysis for the reaction D-xylose + NAD(P)H + H ⁺ ? xylitol + NAD(P)⁺ was carried out and specificity constants (k_cat/K_aldehyde) were measured for the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding capabilities with the substrate binding pocket of the enzyme. dsXR binds NAD(P)⁺ (K_iNAD+ = 70 μM; K_iNADP+ = 55 μM) weakly and NADH (K_i = 8 μM) about as tightly as NADPH (K_i = 14 μM). msXR shows uniform binding of NADPH and NADP ⁺ (K_iNADP+ ≈ K_iNADPH = 20 μM). A quantitative structure-activity relationship analysis was carried out by correlating logarithmic k_cat/K_aldehyde values for dsXR with corresponding logarithmic k_cat/K_aldehyde values for msXR. This correlation is linear with a slope of ≈1 (r² = 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and aldehyde-binding modes are identical in both XR forms. Binary complexes of dsXR-NADH and msXR-NADPH show the same macroscopic pK of ≈9.0-9.5, above which the activity is lost in both enzymes. A lower pK of 7.4 is seen for dsXR-NADPH. Specificity for NADH and greater binding affinity for NAD(P)H than NAD(P)⁺ are thus the main features of enzymic function that distinguish dsXR from msXR.

Full text of DOI:10.1021/jf034426j

7930 J. Agric. Food Chem. 2003, 51, 7930−7935
Multiple Forms of Xylose Reductase in Candida intermedia:
Comparison of Their Functional Properties Using Quantitative
Structure−Activity Relationships, Steady-State Kinetic Analysis,
and pH Studies
#
BERND NIDETZKY,*,‡ KASPAR BRU¨ GGLER,# REGINA KRATZER,‡ AND PETER MAYR
Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria,
and Institute of Food Technology, University of Agricultural Sciences,
Muthgasse 18, A-1190 Vienna, Austria
The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is
NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer
NADH ≈4-fold over NADPH (dual specific xylose reductase; dsXR). To compare the functional
properties of the isozymes, a steady-state kinetic analysis for the reaction D-xylose + NAD(P)H +
H+ T xylitol + NAD(P)+ was carried out and specificity constants (kcat/Kaldehyde) were measured for
the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding
capabilities with the substrate binding pocket of the enzyme. dsXR binds NAD(P)+ (KiNAD+ ) 70 µM;
KiNADP+ ) 55 µM) weakly and NADH (Ki ) 8 µM) about as tightly as NADPH (Ki ) 14 µM). msXR
shows uniform binding of NADPH and NADP+ (KiNADP+ ≈ KiNADPH ) 20 µM). A quantitative structure-
activity relationship analysis was carried out by correlating logarithmic kcat/Kaldehyde values for dsXR
with corresponding logarithmic kcat/Kaldehyde values for msXR. This correlation is linear with a slope of
≈1 (r 2 ) 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and
aldehyde-binding modes are identical in both XR forms. Binary complexes of dsXR-NADH and
msXR-NADPH show the same macroscopic pK of ≈9.0-9.5, above which the activity is lost in both
enzymes. A lower pK of 7.4 is seen for dsXR-NADPH. Specificity for NADH and greater binding
affinity for NAD(P)H than NAD(P)+ are thus the main features of enzymic function that distinguish
dsXR from msXR.
KEYWORDS: Isozymes; dual coenzyme specificity; binding affinity; xylose utilization; xylose fermentation
INTRODUCTION
balanced in respect to the redox cofactors involved. Conse-
quently, formation of fermentation byproducts such as glycerol
and xylitol occurs and reflects the need for balancing the pools
of NAD+/NADH and NADP+/NADPH, respectively. Obvi-
ously, this will lower ethanol yield from xylose (reviewed in
ref 1). Therefore, NADH-linked activity of XR is an important
physiological and technological requirement for efficient yeast
xylose fermentation (1 and references cited therein). If XR used
NADH rather than NADPH, cofactor imbalances during xylose
conversion under oxygen-limited conditions might be alleviated
substantially.
Biochemical work from different laboratories has shown that
yeast XRs can be classified according to coenzyme specificity
into a group of NADPH-specific reductases (monospecific XR;
msXR) and another enzyme group active with NADPH and
NADH (dual specific XR; dsXR) (4; reviewed in refs 6 and 7).
dsXR is present in all yeast taxae known to ferment xylose such
as Pichia stipitis, Candida shehatae, Candida tenuis, and
Pachysolen tannophilus (1 and references cited therein). Both
msXR and dsXR are homodimers composed of identical
subunits of ≈36 kDa molecular mass (4, 6, 7). The available
D-xylose is the main constituent monosaccharide of plant
hemicellulose. The vast amounts of hemicellulose-containing
raw materials accumulating as process wastes in agriculture and
forestry make D-xylose a potential source for production of
large-volume commodities such as fuel ethanol (1). Yeast
fermentation of D-xylose has therefore attracted considerable
interest in biotechnological research (1-3). The initial catabolic
pathway for xylose in yeast is contributed by two oxidoreduc-
tases, xylose reductase [XR; alditol:NAD(P)+ 1-oxidoreductase;
EC 1.1.1.21] and xylitol dehydrogenase (XDH; alditol:NAD+
2-oxidoreductase; EC 1.1.1.9) (1-3). XR catalyzes the first step
by reducing the C1 carbonyl group of xylose, yielding xylitol
as the product (4). Xylitol is then oxidized by XDH to give
D-xylulose (5). Because XDH requires NAD+ whereas XR
generally prefers NADPH over NADH, the pathway is not
* Corresponding author (telephone +43-316-873-8400; fax +43-316-
873-8434; e-mail bernd.nidetzky@tugraz.at).
‡ Graz University of Technology.
# University of Agricultural Sciences.
10.1021/jf034426j CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/02/2003

Multiple Forms of Candida intermedia Xylose Reductase
J. Agric. Food Chem., Vol. 51, No. 27, 2003 7931
respectively; KiA is the apparent dissociation constant of the enzyme-
coenzyme complex (µM); and KA and KB are Michaelis constants (µM;
mM) for A and B, respectively. Inhibitor binding studies in which
NAD(P)+ and NAD(P)H served as product inhibitors of, respectively,
the enzymic reduction of xylose and oxidation of xylitol were carried
out essentially as described by Neuhauser et al. (10). Initial rates were
fitted to the Michaelis-Menten equation expanded as required to
incorporate inhibition by a competitive, mixed-type, or uncompetitive
inhibitor (10).
pH studies were carried out in the direction of NAD(P)H-dependent
xylose reduction. Experiments were performed in 50 mM potassium
phosphate buffer (pH range 5.0-7.0) and 50 mM Tris/HCl (pH range
g7.0-9.5). Initial rates were acquired under conditions in which
[xylose] was varied and [NADH] or [NADPH] was constant and
saturating (>5 KNAD(P)H). pH profiles of log kcat and log(kcat/Kxylose) that
were level below pKb and decreased above pKb were fitted to eq 3. pH
profiles were fitted to eq 4 when log kcat or log(kcat/Kxylose) decreased
both below pKa and above pKb.
sequence information for XR enzymes that have been character-
ized at the protein level (6, 7) indicates that primary structures
of msXR and dsXR share a quite high entire-chain similarity
(≈70%). To understand their relative roles in xylose fermenta-
tion, the functional properties of dsXR and msXR must be
known in detail.
We report here a detailed comparison of msXR and dsXR
from the xylose-fermenting yeast Candida intermedia. This yeast
system was chosen because, first of all, C. intermedia is a good
natural producer of ethanol from xylose (8). C. intermedia msXR
and dsXR activities are induced in the presence of D-xylose
and repressed in the presence of D-glucose (P.M. and B.N.,
unpublished results), emphasizing a common major function of
the isozymes in the metabolism of xylose. Second, there is the
considerable advantage that msXR and dsXR occur at the same
time in a single organism. A rigorous comparative evaluation
of XR isoforms is thus possible in the absence of complexity
due to different microbial species, one producing msXR and
another producing dsXR (4). Third, a preliminary characteriza-
tion of C. intermedia dsXR has revealed that unlike other dsXRs
purified and characterized biochemically so far, this enzyme
prefers NADH over NADPH (9). Although there is good
evidence that NADPH-preferring dsXR can effectively utilize
NADH, it is not completely clear to what extent this occurs in
vivo. This reinforces the potential utility of a NADH-preferring
dsXR.
Our comparison is based on (i) a full steady-state kinetic
analysis for each isozyme in regard to the natural, NADPH- or
NADH-dependent reaction with D-xylose, (ii) a detailed char-
acterization of the substrate specificities of the two XR isoforms
coupled to a quantitative structure-activity relationship analysis
through which we measure the similarity between the aldehyde
binding pockets of msXR and dsXR, and (iii) pH effects on
the enzymic reduction of xylose. Therefore, functional properties
of msXR and dsXR are examined in respect to enzyme
interactions with both the substrate and the coenzyme, and a
comprehensive picture results.
logY ) log[Ymax/(1 + Kb/[H+])]
(3)
(4)
logY ) log[Ymax/(1 + [H+]/Ka + Kb/[H+])]
In eqs 3 and 4, Y is kcat or kcat/Kxylose, Ymax is the maximum value of Y,
Ka and Kb are macroscopic dissociation constants, and [H+] is the proton
concentration. Suitable control experiments showed that msXR and
dsXR were stable in the chosen pH range over the time of the assay
(<5 min). Initial-rate measurements in the overlapping pH range of
Tris and phosphate buffer (pH 7-8) revealed clearly that the buffer is
not a source of observable pH dependencies of the kinetic parameters.
RESULTS AND DISCUSSION
Kinetic Characterization of D-Xylose Reduction by msXR
and dsXR. To provide a basic comparison of msXR and dsXR
with regard to their function in D-xylose reduction (eq 1), we
carried out a full steady-state kinetic analysis for each isozyme
and measured initial rates for NAD(P)H-dependent reduction
of D-xylose and NAD(P)+-dependent oxidation of xylitol at 25
°C and pH 7.0. Two separate sets of kinetic experiments were
conducted with dsXR using either NADP(H) or NAD(H) as
coenzyme of the enzymic oxidoreduction. Initial rates were
obtained under conditions in which the substrate D-xylose (20-
200 mM) or xylitol (20-940 mM) was varied at five different
levels using four to five constant concentrations of coenzyme
NAD(P)H (10-205 µM) or NAD(P)+ (38-600 µM). In both
reaction directions of eq 1, Lineweaver-Burk plots of the
reciprocal initial rates versus the reciprocal substrate concentra-
tions yielded four to five straight lines, each corresponding to
a single coenzyme concentration, that intersected to the left of
the ordinate axis (not shown). This pattern of the lines is
indicative of a reaction mechanism in which a ternary complex
between enzyme, coenzyme, and substrate must be formed
before the first product is released (10).
In consideration of precedent in the literature (10-13), our
assumption was that the kinetic mechanisms of msXR and dsXR
would likely be ordered such that NAD(P)H binds before
D-xylose and that xylitol is released before NAD(P)+. Deter-
mination of the complete steady-state kinetic mechanism of each
isozyme was, clearly, beyond the goal of this work. However,
a basic series of inhibitor binding studies were carried out with
the aim of supporting the above working hypothesis. For msXR,
NADP+ behaves as a competitive inhibitor against NADPH (Ki
) 25 ( 4 µM). Likewise, NADPH is a competitive inhibitor
against NADP+ (Ki ) 20 ( 5 µM). Competitive inhibition of
NADPH versus NADP+ and vice versa was observed both at
unsaturating (<Ksubstrate; see later) and saturating concentrations
of xylose and xylitol, respectively. An identical inhibition pattern
MATERIALS AND METHODS
Materials. msXR and dsXR from C. intermedia (also named
KluyVeromyces cellobioVorus) were produced and purified to apparent
homogeneity as described recently (9). All used substrates were of the
highest purity available and obtained from Sigma-Aldrich or Fluka.
Kinetic Measurements. Apparent kinetic parameters, that is, the
turnover number kcat (s-1) and the apparent Michaelis constant Kaldehyde
(mM), for the NAD(P)H-dependent reduction of different aldehydes
by msXR and dsXR were determined according to reported methods
(4, 9). If saturation in substrate was not achieved in the experiment
(e.g., because of limited solubility of the aldehyde or weak apparent
binding), the catalytic efficiency (kcat/Kaldehyde) was obtained from the
part of the Michaelis-Menten curve where the initial rate is linearly
dependent on the substrate concentration. Initial rates were recorded
spectrophotometrically under conditions in which the concentration of
aldehyde was varied and NADPH or NADH was present at a constant
and saturating concentration of 220 µM.
A full set of kinetic parameters for the reaction in eq 1 was obtained
D-xylose + NAD(P)H + H+ T xylitol + NAD(P)+
(1)
from initial-rate measurements in the forward direction (xylose reduc-
tion) and reverse direction (xylitol oxidation) at 25 °C and pH 7.0 and
nonlinear fits of data to eq 2
V ) kcat[E][A][B]/{KiAKB + KB[A] + KA[B] + [A][B]} (2)
where V is the initial rate; [E] is the molar concentration of enzyme
subunits (36 kDa) (9); [A] and [B] are molar coenzyme concentrations
[NAD(P)H; NAD(P)+] and substrate concentrations (D-xylose; xylitol),

7932 J. Agric. Food Chem., Vol. 51, No. 27, 2003
Nidetzky et al.
seen for dsXR appears to be well matched to these in vivo
conditions, which could prevail during xylose fermentation by
C. intermedia. The observed KiNADPH/KiNADH ratio of 1.7 in
dsXR is similar to the physiological ratio of the reduced
nucleotides. On the basis of kcatR/(KiNAD(P)HKxylose) values and
assuming that NADPH will act as competitive inhibitor with
respect to NADH and vice versa, we can then estimate that
dsXR-catalyzed xylose reduction under the fermentation condi-
tions will take place using NADPH and NADH at similar rates.
Characterization of the Substrate Binding Sites of msXR
and dsXR. Initial rates of aldehyde reduction by msXR and
dsXR were measured using a series of sugars and related
aldehyde substrates, as shown in Tables 2 and 3. If not stated
otherwise, NADH was used as the coenzyme for dsXR-catalyzed
reactions. However, we carried out control experiments in which
NADPH was employed and reduction of unsubstituted straight-
chain aldehydes by dsXR was measured. The relative substrate
specificity of dsXR was not dependent on the coenzyme used.
The substrate series was chosen such that the nonreacting parts
of the aldehydes displayed a representative variation in respect
to the number of carbon atoms, the extent to which the carbon
chain was substituted with hydroxy groups, and the stereo-
chemical orientation of the hydroxy groups present. It was
expected that if the aldehyde-binding sites of msXR and dsXR
differ significantly in their interactions with the selected
substrates, an isozyme-related pattern of specificity will result
and reveal the key components of individual substrate prefer-
ence.
Apparent kinetic parameters for msXR and dsXR are sum-
marized in Tables 2 and 3, respectively. Unless indicated
otherwise, they were obtained from nonlinear fits of the initial-
rate data to the Michaelis-Menten equation. As required for a
realistic comparison of kcat/Kaldehyde values (4), the experimental
catalytic efficiencies were corrected for the portion of free
aldehyde present in an aqueous solution of the respective aldose
(16) as it is the open-chain free-aldehyde form that is the true
substrate of xylose reductase (4) (cf. Tables 2 and 3). Values
of kcat/Kxylose are identical for msXR and dsXR within the
experimental error of (15%. Hence, by the criterion of catalytic
efficiency both enzymes are equally well qualified for xylose
catabolism. kcat/Kaldehyde values for reduction of “good” substrates
are in the range of 1000-3000 mM-1 s-1 and very similar for
both isozymes. These include D-erythrose (four carbons);
D-xylose and L-arabinose (five carbons); and L-idose and
D-fucose () 6-deoxy-D-galactose) (six carbons). msXR differs
most significantly from dsXR (≈10-fold) in respect to catalytic
efficiencies for reduction of unsubstituted straight-chain alde-
hydes, which are smaller for dsXR (Tables 2 and 3). Another
interesting difference between the two isozymes is borne out
by comparing enzymic reactions with D-glucose. dsXR displays
a ≈10-fold specificity for the reduction of D-xylose over the
reduction of D-glucose (Table 3). By contrast, D-glucose is about
as good a substrate of msXR as is D-xylose (Table 2). Estimates
for the intracellular levels of free D-xylose and D-glucose in C.
intermedia are not available, but concentrations of free D-glucose
in the cytosol are probably low due to the rapid conversion into
D-glucose 6-phosphate, as was shown for the related yeast
system Saccharomyces cereVisiae (17). It would seem unlikely,
therefore, that D-glucose could compete with D-xylose to serve
as a substrate of msXR.
Table 1. Kinetic Parameters for msXR and dsXR at 25 °C and pH 7.0
parameter
msXR
28± 1
77 ± 12
28 ± 4
20 ± 5
360 ± 44
0.6 ± 0.02
480 ± 40
16 ± 3
25 ± 4
1.3 ± 0.06
360
dsXR (NADH)
dsXR (NADPH)
kcatRa (s-1
)
16± 1
16.3 ± 0.5
64±8
K
xylose (mM)
30 ± 8
KNAD(P)H (µM)
30± 6
19± 2.5
14 ± 6.8
250 ± 26
0.8 ± 0.01
160 ±10
31± 3
53 ± 9
5± 0.3
190
K
k
iNAD(P)H (µM)
8.1 ± 0.9
530 ± 114
0.9 ± 0.02
40 ± 5
catR/Kxylose (M-1 s-1
kcatOa (s-1
xylitol (mM)
KNAD(P)+ (µM)
)
)
K
37± 5
K
+ (µM)
74± 22
23 ± 2
iNAD(P)
k
catO/Kxylitol (M-1 s-1
)
b
K
220
eq
a R, aldehyde reduction; O, alcohol oxidation. b Calculated from the experimental
parameters using the Haldane relationship K ) (kcatR/Kxylose)K
+/{(kcatO
/
eq
iNAD(P)
Kxylitol)KiNAD(P)H)}; standard deviations for Keq are e30% of the reported values.
was observed for dsXR using NADH (Ki ) 8 ( 1 µM) and
NAD+ (Ki ≈ 100 µM) as the inhibitors of the reverse and
forward reaction directions, respectively. These data imply the
formation of binary complexes between msXR and NADPH or
NADP+ and between dsXR and NADH or NAD+, as required
by the ordered mechanism in which coenzyme binds first and
substrate second. The apparent dissociation constants of the
binary complexes are given by the Ki values.
Initial-rate data for D-xylose reduction and xylitol oxidation
were fitted to eq 2 with the assumption of an ordered kinetic
mechanism. Results are summarized in Table 1. The internal
consistency of the kinetic parameters was checked by using the
Haldane relationship, as shown in the footnotes of Table 1. The
calculated values of between 190 and 370 for the equilibrium
constant (Keq) at pH 7.0 and 25 °C compare reasonably to an
experimentally observed value of ≈400 ((20%) under these
conditions. Ki values for NADP(H) and NAD(P)+ thus obtained
(Table 1) agree well with Ki values derived from inhibition
experiments.
The data in Table 1 provide a basic set of kinetic parameters
of msXR and dsXR needed for a comparative analysis of
isozyme function. On the basis of the expression kcatR
/
(KiNAD(P)HKxylose), we conclude that dsXR prefers NADH 3.6-
fold over NADPH. Considering data in the literature indicating
a 5-10-fold preference of dsXR for NADPH over NADH
(reviewed in refs 1, 6, and 7; 10), this is an unusually high and
thus unique specificity for NADH! Jeffries and co-workers
reported XR activities in the yeast Candida boidinii that were
higher with NADH than NADPH as cosubstrate (14). However,
the C. boidinii XR system has not been explored biochemically.
Because kcatR/Kxylose is much greater (>20-fold) than kcatO/Kxylitol
in all shown cases, we do not consider oxidation of xylitol to
be a relevant reaction catalyzed by msXR or dsXR under any
physiological boundary conditions. Comparison of KiNADPH and
KiNADP+ values for msXR indicates that NADPH and NADP+
bind to this enzyme with nearly identical apparent affinities.
Now, the ratio of the intracellular concentrations of NADPH
and NADP+ in sugar-metabolizing yeast will always be such
that NADPH is present in excess (15). Therefore, msXR will
generally be primed for reductive metabolism of xylose, and
product inhibition by NADP+ will not be significant. An
analogous conclusion can be drawn for dsXR utilizing NADPH,
considering the KiNADPH/KiNADP+ ratio of 3.8. An interesting
result for dsXR is the 9-12-fold tighter apparent binding of
NADH than NAD+ (KiNAD(H)). The characteristic ratios of
NADH/NAD+ and NADPH/NADH in glucose-limited anaerobic
culture of baker’s yeast are approximately 0.15 and 2.8,
respectively (15). Arguing by analogy to S. cereVisiae because
data for C. intermedia are not available, the KiNADH/KiNAD+ ratio
For both isozymes, the value of kcat/Kaldehyde displays a strong
dependence on the structure of the nonreacting substrate part,
particularly the presence and configuration of hydroxy groups.
In the case of msXR, the variation in catalytic efficiency across
the series of pentoses and hexoses in Table 2 is up to

Multiple Forms of Candida intermedia Xylose Reductase
J. Agric. Food Chem., Vol. 51, No. 27, 2003 7933
Table 2. Apparent Kinetic Parameters of msXR for NADPH-Dependent Reduction of Polyhydroxylated and Other Aldehydesa
c
#
kcat/K
−∆∆G of
aldehyde
(mM-1s-1
b
substrate
%
K
aldehyde
c (µM)
kcat (s-1
)
)
kcat/Kaldehyde (kJ/mol)
DL-glyceraldehyde
propionaldehyde
D-erythrose
24.25
1140
24.3
6.9
24.3
21.2
21.3
0.09
736
0.93
13.5
0.0
16.5
0.0
100
78000
33
4
100
butanal
23000
D-xylose
0.02
16
23.5
1470
1.4d
134
36.8
1230
157
5.31
13.9
−3.3
8.0
D-lyxose
0.03
0.05
0.01
0.03
0.03
D-ribose
91
38
20
117
3900
12.2
1.4
24.5
18.4
20.7
2-deoxy-D-ribose
L-arabinose
L-lyxose
4.8
13.5
8.4
0.0
pentanal
100
L-idose
0.1
3260d
1370
117
249
38
14.5
12.3
6.2
D-glucose
0.0024
0.008
0.02
6
58
8.2
6.8
15.2
8.4
20.7
2-deoxy-D-glucose
D-galactose
2-deoxy-D-galactose
D-fucose
D-mannose
hexanal
61
8.1
0.03
0.007
0.005
221
10
3.4
2070
13.3
5.6
92d
100
9.47d
0.0
#
a The NADPH concentration was 220 µM and saturating. Initial rates were determined in 50 mM potassium phosphate buffer, pH 7.0, at 25 °C. ∆∆G was calculated
according to eq 5. Standard deviations were <15%. b Content of free aldehyde in solution (16). c
aldehyde in solution. d Obtained from the linear part of the Michaelis−Menten curve.
Kaldehyde and kcat/Kaldehyde are corrected for the available portion of free
Table 3. Apparent Kinetic Parameters of dsXR for NADH-Dependent Reduction of Polyhydroxylated and Other Aldehydesa
c
#
kcat/K
−∆∆G of
aldehyde
(mM-1 s-1
b
substrate
%
K
aldehyde
c (µM)
kcat (s-1
)
)
kcat/Kaldehyde (kJ/mol)
DL-glyceraldehyde
propionaldehyde
D-erythrose
24.25
2425
14.1
4.6
27.5
5.4
5.8
12.7
0.0
22.4
0.0
100
132000
20
0.035
4
100
1380
butanal
33000
0.16
D-xylose
D-lyxose
D-ribose
L-arabinose
L-lyxose
pentanal
0.02
10
16.9
1690
20.7
5.2
12.9
17.7
11.8
0.0
3.3d
72
0.05
0.03
0.03
68
28
144
4.9
13.5
6.6
482
46
0.40
100
14700
5.9
L-idose
0.1
1820d
170
36d
19.5
13.6
9.8
16.9
9.2
19.2
9.0
0.00
D-glucose
0.0024
0.008
0.02
33
5.6
2-deoxy-D-glucose
D-galactose
2-deoxy-D-galactose
D-fucose
D-mannose
hexanal
15
126
7
9.4
3.5
10.2
627
28
0.03
0.007
0.005
1457
28d
100
0.69d
#
a The NADH concentration was 220 µM and saturating. Initial rates were determined in 50 mM potassium phosphate buffer, pH 7.0, at 25 °C. ∆∆G was calculated
according to eq 5. Standard deviations were <15%. b Content of free aldehyde in solution (16). c
solution. d Obtained from the linear part of the Michaelis−Menten curve.
Kaldehyde and kcat/Kaldehyde are corrected for the portion of free aldehyde in
1000-fold. Aside from variations in apparent Kaldehyde value (37-
fold), this reflects a significant variation in kcat (up to 17-fold).
The change of kcat/Kaldehyde across essentially the same series of
substrates is ≈512-fold for dsXR. The observed variation in
kcat is also smaller for dsXR than it is for msXR (4.5-fold). A
strong dependence of kcat on aldehyde side-chain structure could
occur if catalysis was a major rate-limiting step of the reaction
of msXR. Poor substrates react slowly, and so both turnover
number and catalytic efficiency will be small. In an alternative
scenario, aldehydes are bound to msXR in two different ways,
one that is productive and promotes the reaction and another
that leads to a catalytically incompetent complex which must
dissociate to regenerate the active enzyme bound to NADPH.
If nonproductive binding takes place with certain substrates,
the observed values of kcat for reduction of these aldehydes will
be smaller than they would be if the reactions occurred
exclusively through productive pathways. In any case, the value
of kcat/Kaldehyde represents an extrapolation to an infinitely small
substrate concentration, that is, conditions in which nonproduc-
tive binding cannot occur. Therefore, it will always be an
accurate indication of the catalytic efficiency. The kcat values
of msXR for reduction of straight-chain aldehydes are high (≈20
s-1), whereas, at the same time, the corresponding specificity
constants are among the lowest observed for this enzyme.
Therefore, this suggests that nonproductive binding may occur
during the reaction of msXR with certain aldehydes.
Comparison of the catalytic efficiency for reduction of a
selected aldose with the catalytic efficiency for reduction of an
unsubstituted straight-chain aldehyde displaying an equivalent
number of carbon atoms (cf. Tables 2 and 3) is relevant. It can
provide an estimate of the net contribution of the hydroxy groups
in this aldose to the specificity of the enzymic reaction. We
employ eq 5 to relate a ratio of two specificity constants to a
differential Gibbs free energy that is derived from bonding with

7934 J. Agric. Food Chem., Vol. 51, No. 27, 2003
Nidetzky et al.
Figure 2. pH profiles of log kcat/Kxylose for C. intermedia XR isozymes:
(b) dsXR (NADH as coenzyme); (O) dsXR (NADPH as coenzyme); (1)
Figure 1. Quantitative structure−activity relationship analysis for the
NADPH-dependent reduction of aldehydes by msXR and the NADH-
dependent reduction of the same substrates by dsXR. The figure shows
msXR (NADPH as coenzyme). kcat/K
values were obtained from
aldehyde
initial-rate data recorded in the presence of saturating coenzyme levels
(200−250 µM) and are not corrected for the portion of free aldehyde in
aqueous solutions of D-xylose. The solid lines are fits of the data to eq
3 or 4, as indicated in Table 4.
a log−log correlation of kcat/K
values for msXR and dsXR. kcat/
aldehyde
Kaldehyde values are taken from Tables 2 and 3. The solid line shows the
fit of the data to a straight line.
the available hydroxy groups and used for transition-state
stabilization
Table 4. pK Values from pH Profiles of log kcat/Kxylose for msXR and
dsXR
∆∆G# ) - RT ln(kcat/Kaldose/kcat/Kstraight-chain aldehyde) (5)
enzyme (coenzyme)
eq fitted
pK
a
pK
b
Ymax (M-1 s-1
)
where R is the gas constant in J K-1mol-1 and T is the
temperature in K. The calculated ∆∆G# values are found in
Tables 2 and 3. Maximum transition state stabilization energies
of 14 and 19-20 kJ/mol have been estimated for reactions
catalyzed by msXR and dsXR, respectively. This result could
imply that the catalytic efficiency of dsXR depends slightly more
[≈6 kJ/mol () 20 - 14)] on bonding interactions with sugar
hydroxyls than that of msXR.
msXR (NADPH)
dsXR (NADH)
dsXR (NADPH)
4
3
3
5.5 ± 0.1
9.0 ± 0.2
9.5 ± 0.5
7.4 ± 0.1
237 ± 21
331 ± 39
302 ±36
corresponding specificity constants of dsXR using NADH as
cosubstrate. This figure displays a simple comparative analysis
of quantitative structure-activity relationships for dsXR and
msXR. Its value is that it can measure similarities in the
substrate-binding pockets of both enzymes or detect the absence
of such similarities. Figure 1 clearly suggests a relationship
between the two sets of data that is linear overall. The data
were fitted to a straight line, as shown in the figure, and a slope
value of 1.10 and a correlation coefficient (r2) of 0.912 were
obtained from the linear fit. The interpretation of the structure-
activity analysis is that binding modes for sugars are virtually
identical at the substrate binding sites of msXR and dsXR.
pH Effects on Xylose Reduction by msXR and dsXR.
Comparative pH studies for related enzymes catalyzing the same
reaction can reveal subtle differences in pH-dependent ionization
of groups involved in substrate binding and catalysis. Deter-
mination of pH effects on xylose reduction by msXR and dsXR
was therefore considered to be potentially valuable. Apparent
kinetic parameters for NADPH-dependent reduction of D-xylose
by msXR and dsXR were obtained in the pH range of 5.0-9.5.
The data in Tables 2 and 3 also show that 2-deoxy derivatives
of sugars are generally much poorer substrates than the
corresponding parent compounds (D-ribose, D-galactose, and
D-glucose), all of which display a C-2(R) hydroxy group. A
∆∆G# value, which describes the differential binding energy
stabilizing the transition state of the enzymic reaction with the
parent sugar over the transition state for the same reaction with
the corresponding 2-deoxy derivative, can be calculated using
Tables 2 and 3. It is in a range of ≈4-6 kJ/mol. Therefore,
this leads to the interpretation that non-covalent bonding with
an appropriately oriented 2-OH contributes up to 43% of the
maximum value of binding energy in the transition state of
NAD(P)H-dependent aldehyde reduction by msXR and dsXR
(i.e., 6 of 14 kJ/mol). Likewise, comparisons of enzymic
reductions of sugars having R-configured C-2 with reductions
of the corresponding C-2(S) epimers (D-xylose vs D-lyxose;
D-glucose vs D-mannose) indicate that losses of transition-state
stabilization energy in the C-2(S) substrates are almost identical
to those in the 2-deoxy substrates. Therefore, aldose substrates
diplaying an R-configured C-2 are preferred over their C-2
epimers, clearly because the substrate-binding sites provide
favorable (likely hydrogen bonding) interactions with the C-2(R)
hydroxy group, which are lacking in C-2(S) substrates. Non-
covalent enzyme-substrate interactions at C-3 of the aldehyde
are also important, as evident from comparisons of catalytic
efficiencies for reductions of the C-3 epimers D-xylose and
D-ribose or L-arabinose and L-lyxose.
k
cat and kcat/Kxylose values for the NADH-dependent reaction of
dsXR were determined in the pH range of 5.0-10.3. All pH
profiles for log kcat displayed a decrease at high pH that was
either truly biphasic or had a marked hollow (18) in the pH
range of 8.0-9.0 (not shown). pKb values for the pH dependence
of log kcat were, therefore, not determined, and interpretation
of these profiles would be speculative. Plots of log kcat/Kxylose
against pH are shown in Figure 2 and reveal that, in all cases,
activity decreases at high pH. Data were fitted to the appropriate
equation, and results are summarized in Table 4. The pH profile
for dsXR (using NADPH as coenzyme) decreases above an
apparent pKb value of 7.4. Interestingly, when NADH is
employed as the coenzyme, a significantly higher apparent pKb
Figure 1 shows a log-log correlation of kcat/Kaldehyde values
for NADPH-dependent reduction of sugars by msXR with

Multiple Forms of Candida intermedia Xylose Reductase
J. Agric. Food Chem., Vol. 51, No. 27, 2003 7935
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value of 9.5 is observed. In the case of msXR, which obviously
was assayed with NADPH, the pH profile decreases with a (1
slope below an apparent pKa of 5.5 and above another pKb of
9.0. For the interpretation of the observed pH effects we must
consider that NAD(P)H was saturating in the experiments and
D-xylose does not show ionization in the used pH range.
Therefore, the pH profiles of log kcat/Kxylose reflect pH-dependent
ionizations in binary enzyme-coenzyme complexes. Note that
because [coenzyme] was saturating, the difference in pKb value
seen for NADH- and NADPH-linked reactions of dsXR cannot
be attributed to a pH-dependent ionization of the 2′-phosphate
group of free NADPH. The pH profiles reveal macroscopic pKb
values of groups required for D-xylose binding and/or catalysis.
A picture of considerable similarity among XR isoforms
emerges from the comparison of pH effects in Figure 2 and
Table 4; particularly, the pKb values for msXR-NADPH and
dsXR-NADH are the same within standard error. The lower
pKb seen in dsXR-NADPH, compared to dsXR-NADH, is
not directly open to interpretation. However, the marked
difference in pKb could be explained if the ionizable groups
showing in the two binary complexes were not the same or if
the pK of the functional group(s) was in some way dependent
on the 2′-phosphate group of NADPH. The intracellular pH of
C. intermedia is not known. However, if we draw again analogy
to baker’s yeast and assume a probable in vivo pH value of 6.8
(19), msXR-NADPH and dsXR-NADH complexes are fully
protonated and hence in the correct ionization state for xylose
reduction. Under the assumed pH conditions, the dsXR-
NADPH complex could be partly ineffective catalytically due
to noticeable deprotonation. If that was truly the case, pH effects
could contribute to a discrimination of dsXR against the use of
NADPH.
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Conclusions. Aside from the ability to utilize NADH in place
of NADPH, the functional feature that distinguishes dsXR most
from msXR is differential binding recognition of NADH versus
NAD+. The aldehyde-binding sites of the isozymes are very
similar in regard to the non-covalent interactions that they can
provide with a range of substrates and the pH-dependent
ionization of functional groups on the protein. The physiological
implication of binding NADH more tightly than NAD+ is
without doubt alleviating product inhibition by NAD+, which
will be present in excess over NADH at all times, even when
during xylose fermentation respiratory chain-linked reoxidation
of NADH is low. The fact that dsXR has a ≈4-fold higher
specificity for NADH than NADPH could make this enzyme
an interesting novel candidate to be used in metabolic engineer-
ing of the yeast xylose metabolism, likely in S. cereVisiae
(1, 2). We emphasize that this is the first XR enzyme found to
prefer NADH. Obviously, increased levels of dsXR activity
could contribute to an improvement of ethanol production from
D-xylose by reducing the cofactor imbalance of the initial
catabolic pathway.
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Xylose fermentation by yeasts. 4. Purification and kinetic studies
of xylose reductase from Pichia stipitis. Appl. Microbiol.
Biotechnol. 1988, 29, 148-154.
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and key enzyme activities in Candida boidinii under different
oxygen transfer rates. J. Ferment. Bioeng. 1995, 80, 513-516.
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J.; Hahn-Ha¨gerdal, B.; Kielland-Brandt, M. C. Expression of the
Escherichia coli pntA and pntB genes, encoding nicotinamide
nucleotide transhydrogenase, in Saccharomyces cereVisiae and
its effect on product formation during anaerobic glucose
fermentation. Appl. EnViron. Microbiol. 1999, 65, 2333-2340.
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Current aspects. AdV. Carbohydr. Chem. Biochem. 1991, 49, 19-
35.
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In vivo analysis of metabolic dynamics in Saccharomyces
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1997, 55, 305-316.
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1977, 45, 273-387.
ABBREVIATIONS USED
XR, xylose reductase; dsXR, dual NADH/NADPH-specific
XR; msXR, NADPH-specific (monospecific) XR; XDH, xylitol
dehydrogenase.
(19) Shanks, J. V.; Bailey, J. E. Estimation of intracellular sugar
concentrations in Saccharomyces cereVisiae using 31P nuclear
resonance spectroscopy. Biotechnol. Bioeng. 1988, 32, 1138-
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LITERATURE CITED
(1) Hahn-Ha¨gerdal, B.; Wahlbom, C. F.; Gardonyi, M.; van Zyl,
W. H.; Cordero Otero, R. R.; Jonsson, L. J. Metabolic engineer-
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Received for review April 25, 2003. Revised manuscript received
October 10, 2003. Accepted October 28, 2003. Financial support from
the Austrian Science Funds (FWF Project P-15208-MOB) and from
the European Commission (EU FAIR CT 96-1098) is gratefully
acknowledged.
JF034426J