From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: A mechanistic study of mannitol 2-dehydrogenase from pseudomonas fluorescens

Article abstract of DOI:10.1074/jbc.M110.108993

Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD⁺-mannitol (K_int) was altered dramatically (10⁴- to 10 ⁵-fold) from being balanced in the wild-type enzyme (K_int ≈ 3) to favoring enzyme-NAD⁺-mannitol in the single site mutants, N191D and N300D. The change in K_int reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD⁺-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pKvalues of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn → Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis.

Full text of DOI:10.1074/jbc.M110.108993

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 40, pp. 30644–30653, October 1, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
From Alcohol Dehydrogenase to a “One-way” Carbonyl
Reductase by Active-site Redesign
A MECHANISTIC STUDY OF MANNITOL 2-DEHYDROGENASE FROM PSEUDOMONAS
□
S
FLUORESCENS
Received for publication, January 29, 2010, and in revised form, July 15, 2010 Published, JBC Papers in Press, July 16, 2010, DOI 10.1074/jbc.M110.108993
‡
‡§1
Mario Klimacek and Bernd Nidetzky
‡
From the Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria
and ACIB GmbH, Petersgasse 14, A-8010 Graz, Austria
§
Directional preference in catalysis is often used to distinguish ditions. However, the directional preference of biological catal-
alcohol dehydrogenases from carbonyl reductases. However, ysis, expressed as the ratio of reaction rate constants in
the mechanistic basis underpinning this discrimination is weak. direction of carbonyl reduction (k ) and alcohol oxidation (k ),
R
O
In mannitol 2-dehydrogenase from Pseudomonas fluorescens, is not uniform among natural enzymes and underpins a widely
stabilization of (partial) negative charge on the substrate oxya- used classification that considers “alcohol dehydrogenases”
nion by the side chains of Asn-191 and Asn-300 is a key feature (k /k Յ 1) and “carbonyl reductases” (k /k ӷ 1). Horse liver
R
O
R
O
of catalysis in the direction of alcohol oxidation. We have dis- alcohol dehydrogenase and human aldose reductase are repre-
rupted this ability through individual and combined substitu- sentative members of each class, having k /k values of ϳ1 (1)
R
O
tions of the two asparagines by aspartic acid. Kinetic data and and 217 (2), respectively. Work on lactate dehydrogenase has
their thermodynamic analysis show that the internal equilib- shown that k /k is sensitive to active-site structural changes
R
O
؉
rium of enzyme-NADH-fructose and enzyme-NAD -mannitol resulting from site-directed mutagenesis (3). However, a clear
4
5
(
K ) was altered dramatically (10 - to 10 -fold) from being bal- mechanistic basis for the distinction between dehydrogenases
int
anced in the wild-type enzyme (K ≈ 3) to favoring enzyme- and reductases is currently lacking.
int
؉
NAD -mannitol in the single site mutants, N191D and N300D.
The oxyanion binding pocket of PfM2DH (Pseudomonas
2
The change in K reflects a selective slowing down of the man- fluorescens mannitol 2-dehydrogenase) constitutes a distinct
int
nitol oxidation rate, resulting because Asn 3 Asp replacement structural motif of proficient catalytic function as an alcohol
(
i) disfavors partial abstraction of alcohol proton by Lys-295 in a dehydrogenase (EC 1.1) (4). The enzyme is a well characterized
step preceding catalytic hydride transfer, and (ii) causes stabili- member of the large and diverse superfamily of long-chain
؉
ϩ
zation of a nonproductive enzyme-NAD -mannitol complex. dehydrogenases and reductases (5, 6). It utilizes NAD to oxi-
N191D and N300D appear to lose fructose binding affinity due dize the C-2 alcohol of mannitol into the keto-group of the
to deprotonation of the respective Asp above apparent pK values open-chain free-carbonyl form of D-fructose (Fig. 1A). Active-
of 5.3 ؎ 0.1 and 6.3 ؎ 0.2, respectively. The mutant incorporat- site preorganization in PfM2DH resulting from the side chains
ing both Asn3Asp substitutions behaved as a slow “fructose of Asn-191 and Asn-300 strongly resembles the well known
reductase” at pH 5.2, lacking measurable activity for mannitol catalytic “oxyanion holes” in serine proteases (4, 7–10). The
oxidation in the pH range 6.8–10. A mechanism is suggested in x-ray crystal structure of PfM2DH bound with mannitol and
which polarization of the substrate carbonyl by a doubly proto- NAD(H) shows that the oxyanion binding site belongs to a
nated diad of Asp and Lys-295 facilitates NADH-dependent strikingly symmetric arrangement of groups, which include the
reduction of fructose by N191D and N300D under optimum pH reactive centers on the substrates and the catalytic residues on
conditions. Creation of an effectively “one-way” reductase by the enzyme, as depicted in Fig. 1B (11).
active-site redesign of a parent dehydrogenase has not been pre-
Mutational analysis has delineated catalytic roles for Asn-
viously reported and holds promise in the development of car- 191 and Asn-300 during NAD(H)-dependent interconversion
bonyl reductases for application in organic synthesis.
of mannitol and fructose by PfM2DH (4, 12). Positioning effects
and electrostatic stabilization are exploited to facilitate forma-
tion of the reactive conformers in each direction of the reaction
Enzymatic NAD or NADP-dependent interconversion of and to provide selective rate enhancement to the hydride trans-
alcohol and carbonyl groups is a key chemical transformation in fer step. For mannitol oxidation, the “oxyanion hole” brings
ϩ
3
biology. The thermodynamic equilibrium constant of the reac- about pK depression in both the ⑀-NH group of the catalytic
a
tion usually favors alcohol under a wide range of external con- base Lys-295 and the reactive hydroxyl group of the substrate.
Deprotonation of the lysine is required to “activate” the dehy-
□S
The on-line version of this article (available at http://www.jbc.org) contains drogenase function of PfM2DH at neutral pH where Lys-295
supplemental text, equations, Tables S1 and S2, Figs. S1–S4, and
references.
1
2
To whom correspondence should be addressed: Institute of Biotechnology
andBiochemicalEngineering, Petersgasse12/I, GrazUniversityofTechnol-
ogy, A-8010 Graz, Austria. Tel.: ϩ43-316-873-8400; Fax: ϩ43-316-873-
The abbreviations used are: PfM2DH, mannitol 2-dehydrogenase from P.
fluorescens; KIE, kinetic isotope effect; MD, molecular dynamics; MES,
4-morpholineethanesulfonic acid; Tris, tris(hydroxymethyl)aminometh-
2
8
434; E-mail: bernd.nidetzky@tugraz.at.
ane; NADD, 4S-[ H]NADH.
3
0644 JOURNAL OF BIOLOGICAL CHEMISTRY
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From Dehydrogenase to Reductase by Active-site Redesign
reductase protein superfamily where some enzymes (family
1
D) utilize the (protonated) side chain of a Glu for strong car-
bonyl group polarization during double-bond reduction in ␣,␤-
unsaturated ketone substrates (15–17).
Herein, we report on a detailed kinetic and mechanistic char-
acterization of three mutants of PfM2DH: N191D, N300D, and
a double mutant incorporating both Asn 3 Asp substitutions
(
N191D-N300D). Using free-energy profile analysis, we com-
pared reactions catalyzed by N191D and N300D to the reaction
catalyzed by the wild-type enzyme. The results reveal that K
int
for both single-site mutants decreased by about 5 orders of
magnitude relative to the corresponding internal equilibrium
for native PfM2DH. Experimental findings are combined with
evidence from molecular dynamics (MD) simulations to sug-
gest that the dramatic change in directional preference toward
fructose reduction results, because replacement of either Asn
by Asp (i) disfavors precatalytic “activation” of enzyme-bound
mannitol by partial deprotonation and, furthermore, (ii)
appears to go along with gain of carbonyl group polarization
ability. The double mutant provided affirmation, because it
lacked measurable activity for mannitol oxidation and behaved
as a slow fructose reductase at low pH where the introduced
Asp residues should be partially protonated. Conversion of an
alcohol dehydrogenase into a carbonyl reductase by rational
redesign of the enzyme active site has to our knowledge not
been previously reported.
EXPERIMENTAL PROCEDURES
FIGURE1. Thereactioncatalyzedby PfM2DH(A)andclose-upstructureof
the enzyme active site (B). X-ray coordinates for enzyme bound to NAD
and mannitol were used in B (PDB entry: 1M2W). The symmetric arrangement
ofthecatalyticgroupsandthereactivesubstratehydroxylisindicatedingray.
Dashed lines show distances Յ 4.1 Å.
ϩ
Chemicals and other materials were described elsewhere (4,
1
2–14). Reported methods were used for preparation of wild-
type PfM2DH (18).
Preparation of N191D, N300D, and N191D-N300D—Inverse
ϩ
with a pK of 9.2 in the enzyme-NAD complex would other- PCR using suitable oligonucleotide primer pairs (N191D, [5Ј-
a
wise be ionized (13). Partial abstraction of the mannitol proton TGCGATGACCTGCCCCACAAT-3Ј] and [5Ј-GGACATCA-
by Lys-295 is thought to be a key catalytic event that primes the CGGTAAACGC-3Ј]; N300D, [5Ј-TTGCTCGACGGCAGCC-
ternary complex for cleavage of the substrate C–H bond (13, ACCTG-3Ј] and [5Ј-GCCGATCTTCATCTCTTCATA-3Ј])
1
4). In the direction of fructose reduction, the oxyanion hole was employed to introduce the desired mutations. In the shown
assists in the chemical conversion with NADH by polarizing the primers, the underlined codons produce the respective site-
reactive carbonyl group of the substrate (4).
directed substitution, and the mismatched bases are indicated
Partial disruption of the oxyanion hole through individual in bold. Plasmid pETR241-PfM2DH encoding C-terminally
site-directed substitution of Asn-191 and Asn-300 by Ala His-tagged wild-type enzyme was used as template for PCR
3
caused a large (Յ10 -fold) decrease in the rate constant for the according to a reported protocol (12). To obtain the gene
overall chemical step (k ) in each direction of the reaction (4). encoding N191D-N300D, we subjected the plasmids for
obs
Expressing the internal equilibrium between enzyme-NADH- N191D and N300D to a double digestion by NcoI and BamHI.
ϩ
fructose and enzyme-NAD -mannitol (K ) as the ratio of k
The NcoI-BamHI fragment containing the relevant mutations
int
obs
in oxidation and reduction, literature data can be used to show for N300D was subcloned into the vector for N191D. The full
(
see supplemental Fig. S1) that mutation of either Asn intro- sequence of each mutated gene was confirmed. All mutants
duces a pH dependence to K not present in the wild-type were produced in Escherichia coli JM109 and purified by known
int
enzyme. With the suggestion, from supplemental Fig. S1, that methods (18). Supplemental Fig. S2 shows that each mutant
K
is sensitive to changes in oxyanion hole properties, we con- was isolated as a full-length protein having the expected molec-
int
sidered that active-site net charge perturbation resulting from ular mass of ϳ55 kDa (analysis by SDS-PAGE; supplemental
the site-specific exchange of one Asn by Asp (N191D and Fig. S2A) and that the overall native fold of PfM2DH was not
N300D) might have a large impact on the relative stability of the altered because of the site-directed substitutions (CD spectro-
ternary complexes in PfM2DH. We further hypothesized that a scopic analysis; supplemental Fig. S2B).
remodeled catalytic center of PfM2DH comprising a highly
Kinetic Characterization—Methods for steady-state kinetic
acidic diad of Asp-191 (or Asp-300) and Lys-295 might be suit- characterization were described in previous reports (12–14,
able for function as “fructose reductase.” This design concept 18). The buffers used (each 100 mM) were MES/NaOH (pH
was strongly supported by precedence within the aldo-keto- 5.2–6.8), Tris/HCl (pH 7.1–9.0), and glycine/NaOH (pH 9.0–
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From Dehydrogenase to Reductase by Active-site Redesign
1
0.5). pH dependences of kinetic parameters (k , k
/
vidual substitutions of Asn-191 and Asn-300 by Asp were built
cat
cat
K
) for oxidation and reduction were determined from in this starting model. Protein structures were fully hydrated
substrate
initial rates recorded at varied concentrations of mannitol and using the simple point charge water model in a cubic box. The
fructose while using a constant, saturating (Ն3 ϫ K pH was adjusted to a value of 7. System charges were neutral-
concentration of NAD (2.0 mM at pH 8.0–10.5; 10.0 mM at pH ized assuming 0.1 M NaCl, resulting in a total model size of
)
coenzyme
ϩ
6
.8–7.5) and NADH (200 ␮M), respectively. Kinetic isotope ϳ22,000 atoms. Energy minimization was done using the steep-
2
effects (KIEs) (resulting from deuteration of substrate (2- H- est descents method such that the maximum of force on any
2
mannitol) or coenzyme (4S-[ H]NADH, NADD) were mea- atom was less than 1000 kJ, followed by position-restrained
sured using reported methods (4, 13). A nomenclature is used simulation at 300 K for 40 ps to equilibrate the water molecules.
where subscripts O and R in k indicate mannitol oxidation MD simulations were then done for minimally 15 ns at constant
cat
and fructose reduction, respectively. Superscript D in a kinetic temperature (300 K, using the velocity rescaling temperature
D
parameter (e.g. k
) indicates a primary deuterium KIE (19). coupling method) and pressure (1 bar, using the Parrinello-
catO
Rapid mixing stopped-flow experiments were performed as Rahman temperature-coupling method). An integration time
described previously (4, 13). Supplemental Table S1 summa- step of 2 fs was used, and the van der Waals radius and Coulomb
rizes the conditions applied. Time courses of formation and interactions (calculated using particle mesh Ewald method)
consumption of NADH were recorded from absorbance at 340 were computed within cutoffs of 9 Å each. Periodic boundary
nm. The dead-time of the stopped-flow instrument was 1.5–6.0 conditions were applied. Simulation trajectories were analyzed
ms depending on the substrate concentration used. KIEs on with GROMACS to compute the root mean square deviation of
transient rate constants for fructose reduction were obtained atomic positions and atom-atom distances, respectively. Visual
from direct comparison of reactions with NADH and NADD molecular dynamics (24) was used to generate simulation mov-
(
supplemental Table S1).
ies. Representative structure snapshots were saved and visual-
Data Analysis—SigmaPlot 2004 version 9.0. was used for ized using the PyMOL molecular graphics system.
data fitting by linear or non-linear least squares regression.
RESULTS
Equation 1 describes a pH – log(k /K ) profile that shows a
cat
m
decrease with ϩ1 slope below pK and is level at high pH (C).
Structural Characterization of Mutants of PfM2DH, by
Equation 2 describes a wave-like pH dependence where log(k
/
Experiments and MD Simulation—N191D, N300D, and
cat
K ) decreases with Ϫ1 slope above pK from a constant value N191D-N300D were obtained as electrophoretically pure pro-
m
at low pH (C ) to a lower constant value at high pH (C ). K is tein preparations that appeared structurally intact and properly
H
L
ϩ
the proton dissociation constant, and [H ] is the proton con- folded (supplemental Fig. S2). The CD spectroscopic signature
centration. Equation 3 is the rate formula for an Ordered Bi Bi of each mutant was identical with limits of error to that of the
kinetic mechanism where substrate A binds to the enzyme wild-type enzyme (supplemental Fig. S2B), suggesting a highly
prior to substrate B (20). K is the apparent dissociation con- similar distribution of secondary structural elements in the
iA
stant for A. K and K are Michaelis constants for A and B, proteins as isolated. To obtain more detailed information on
A
B
respectively. [E] is the molar enzyme concentration, calculated possible perturbations in native protein structure caused by
from protein absorbance at 280 nm using a value of 55.4 individual substitution of Asn-191 and Asn-300, we compared
Ϫ1
Ϫ1
mM cm
for ⑀
of wild-type and mutated forms of wild-type PfM2DH, N191D, and N300D in MD simulations of
280
PfM2DH (21). Equation 4 was used to fit stopped-flow time the (nonproductive) ternary complex between enzyme, NADH,
courses displaying a biphasic (“burst” kinetic) character. and mannitol. The structural trajectory for each protein was
[
NADH] is the concentration of NADH at time t, ⌸ is [NADH] analyzed using the experimental crystal structure of the wild-
t
consumed in the pre-steady state, k
is the transient rate type enzyme as the reference. Supplemental Fig. S3 shows var-
obsR
constant, k is the steady-state rate, and Y is the initially iation of the calculated root mean square deviation values with
ss
0
applied [NADH] minus ⌸. Reactions using NADD instead of time. The simulation results are interpreted to imply that no
NADH were analyzed identically.
log͑k /K ͒ ϭ log͕C/͑1 ϩ K/͓H ͔͖͒
significant change in the overall fold of each enzyme occurred
over a total simulation time of 25 ns. Moreover, the sampled
simulated structures of each mutant were highly similar to
those obtained for wild-type PfM2DH. Except for conforma-
tional rearrangements of catalytic side chains (Asn-191, Asp-
230, and Asn-300) that are relevant mechanistically and will be
described later, individual Asp replacements of Asn-191 and
Asn-300 did not result in a global structural disruption of the
mannitol binding site as simulated. Supplemental Fig. S4 (A–C)
depicts simulation time courses for relevant atom-atom dis-
ϩ
(Eq. 1)
cat
m
ϩ
ϩ
log͑k /K ͒ ϭ log͕͑C ϩ C ͓͑H ͔/K͒͒/͑1 ϩ ͓H ͔/K͖͒
cat
m
L
H
(
Eq. 2)
v ϭ k ͓E͔͓A͔͓B͔/͑K K ϩ K ͓B͔ ϩ K ͓A͔ ϩ ͓A͔͓B͔͒ (Eq. 3)
cat
iA
B
A
B
Ϫkobst
͓
NADH͔ ϭ ͓⌸͔e
Ϫ k t ϩ Y
(Eq. 4)
t
ss
0
MD Simulations—These were performed with the program tances in the active sites of native PfM2DH, N191D, and
GROMACS 4.0.7 (22) using the GROMOS 53A6 force field N300D. According to the results of MD simulation, the site-
(
23). Crystallographic coordinates for PfM2DH having NADH directed substitutions of Asn-191 and Asn-300 were further-
and mannitol bound (chain A, PDB entry: 1M2W) were used. more fully compatible with accommodation of the nicotina-
All selenocysteines present in the experimental structure were mide moiety of coenzyme in a productive orientation (data not
ϩ
replaced by cysteines, and crystal waters were removed. Indi- shown). The apparent dissociation constant for enzyme-NAD
3
0646 JOURNAL OF BIOLOGICAL CHEMISTRY
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From Dehydrogenase to Reductase by Active-site Redesign
(
K ), as obtained from fluorescence titration analysis at pH apparent pK. The pH profiles of log(k /K
) for N191D
d
catR fructose
1
0.0, was ϳ1 mM for all mutants and unchanged as compared and N300D decreased from a constant value at low pH to a
with the corresponding K of the wild-type enzyme (data not lower constant value at high pH.
d
shown). The results from MD simulation and experiments
Combined substitution of Asn-191 and Asn-300 by Asp
therefore support the suggestion that functional consequences caused complete disruption of “mannitol dehydrogenase”
in N191D and N300D can be interpreted to arise from locally activity below detection limit at pH 10.0. No activity toward
disruptive effects of the respective site-directed substitution.
mannitol was measured across the entire pH range studied in
pH Dependences of Kinetic Parameters for the Mutants—In- Fig. 2. However, N191D-N300D displayed significant fructose
dividual site-directed replacements of Asn-191 and Asn-300 by reductase activity, and this was maximal at the lower end of the
Asp introduce an extra ionizable group in the active site of experimental pH range. As shown in Fig. 2B, log(k /K
)
catR fructose
PfM2DH, and this was expected to bring about a change in of the double mutant decreased linearly, with a slope of ϳϪ1, in
pH-activity dependence for the resulting mutants as compared response to an increase in pH from 5.2 to 7.0. At each point of
with the wild-type enzyme. The pH profiles of logk and measurement in this pH range, the rate of substrate reduction
cat
log(k /K
) for N191D and N300D are displayed in Fig. 2, by N191D-N300D was linearly dependent on the fructose con-
cat substrate
A and B, respectively, where they are compared with the corre- centration (data not shown), indicating that apparent binding
sponding pH profiles for native PfM2DH that were taken from of fructose by the double mutant was very weak. No k
value
catR
our previous reports (13, 18). k
was independent of pH was therefore obtained for N191D-N300D.
catO
(
N300D) or showed only a slight linear increase (N191D) as the
To derive relevant parameters from the experimental pH
pH was raised from 6.8 to 10.5. With both mutants, activity for dependences in Fig. 2, we used non-linear fitting of data with a
oxidation of mannitol was below limit of detection at a pH suitable equation (Equations 1 and 2). Results are summarized
smaller than 6.8. k
of N191D and N300D was likewise inde- in Table 1. The apparent pK in the pH profile of log(k
/
catR
catR
pendent of pH in the range 5.2–7.0. Neither single-site mutant
could be saturated with fructose under conditions of pH Ն 8, to much lower values of 5.3 and 6.3 for N191D and N300D,
precluding determination of a k value in this pH range (see respectively. The magnitude of the pK shift caused by each site-
K
) was changed from a value of 9.2 for wild-type PfM2DH
fructose
catR
Fig. 2A). Fig. 2B shows that the pH profiles of log(k
/
directed substitution is remarkable. Displacement of the pH
catO
K
) for N191D and N300D were level at high pH and dependence for k /K
into the low pH region was fur-
mannitol
catR fructose
decreased with a slope of ϩ1 as the pH was decreased below ther enhanced in the double mutant. It is therefore interesting
that the apparent pK observed in the pH profiles of log(k
/
catO
K
) for the mutants was not (N191D) or only slightly
mannitol
changed (N300D) in comparison to the wild-type reference.
At the optimum protonation state for mannitol oxidation
high pH), N191D and N300D displayed similar catalytic effi-
(
ciencies that were lowered by four orders of magnitude as com-
pared with the corresponding k
/K
value for the wild-
catO mannitol
type enzyme. At low pH where fructose reduction proceeds
optimally, N191D was 90-times more efficient, in k /K
catR fructose
terms, than N300D. Considering the large loss of enzyme effi-
ciency in mannitol oxidation resulting from substitution of
Asn-191 or Asn-300 by Asp, it was striking that k /K
catR fructose
FIGURE 2. pH profiles of logkcat (A) and log(kcat/K) (B) for N191D, N300D,
and N191D-N300D in fructose reduction (open symbols) and mannitol
for N191D at low pH was only 28-fold decreased as compared
oxidation (closed symbols). Published data for wild-type PfM2DH are shown with the corresponding catalytic efficiency for wild-type
from previous studies (13, 18). Circles, squares, triangles (downward), and tri-
PfM2DH. At high pH, both single-site mutants retained a low
angles (upward) show kinetic parameters of N191D, N300D, N191D-N300D,
and native enzyme, respectively. Data in parenthesis are initial rates recorded
at a non-saturating concentration of fructose (2.00 M Ͻ Kfructose).
catalytic efficiency for fructose reduction, as shown in Fig. 2B
and Table 1.
TABLE 1
Results from pH studies for PfM2DH mutants at 25 °C
The squared correlation coefficient for each fit was 0.99 or greater.
Enzyme
Parameter
pK
C
M
Equation used
sϪ1
Ϫ1a
N191D
kcatO/Kmannitol
9.2 Ϯ 0.4
3.2 Ϯ 0.4
(1)
(2)
C ϭ 0.7 Ϯ 0.1
L
4
3
N300D
k
k
k
/K
5.3 Ϯ 0.1
10 Ϯ 1
CH ϭ 1.9 ϫ 10 Ϯ 4 ϫ 10
8 Ϯ 2
catR fructose
/K
(1)
(2)
catO mannitol
/K
6.3 Ϯ 0.2
C ϭ 0.7 Ϯ 0.1
catR fructose
L
C
ϭ 211 Ϯ 40
H
N191D-N300D
Wild-type
k
k
k
/K
Ͻ5.2
slope: Ϫ0.9 Ϯ 0.1
Linear regression
catR fructose
b
4
4
/K
9.2 Ϯ 0.3
5 ϫ 10 Ϯ 2 ϫ 10
catO mannitol
9.3 Ϯ 0.2c
5.3 ϫ 10 Ϯ 0.6 ϫ 10
5
5
/K
catR fructose
a
b
c
C is the pH-independent value of the kinetic parameter analyzed. C
L H
and C are constant values of the respective parameter at low and high pH, respectively.
Data are from Ref. 13.
Data are from Ref. 18.
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From Dehydrogenase to Reductase by Active-site Redesign
TABLE 2
Kinetic parameters for wild-type PfM2DH and mutants thereof
N191D-N300D
pH 10.0
N300D
N191D
Wild type
Parameter
pH 10.0
pH 6.8a
pH 10.0
pH 7.1a
pH 10.0b
pH 7.1a
Mannitol oxidation
Ϫ1
c
Ϫ5
Ϫ4
Ϫ5
5.5 ϫ 10 Ϯ 2 ϫ 10Ϫ5
Ϫ4
k
k
(s
(s
)
–
0.0015 Ϯ 3 ϫ 10
0.0014 Ϯ 3 ϫ 10
0.0011 Ϯ 2 ϫ 10
40 Ϯ 0.5
15.9 Ϯ 0.3
1041 Ϯ 100
21 Ϯ 2
catO
obsO
Ϫ1
c
d
d
)
–
k
k
k
k
1483 Ϯ 80
0.40 Ϯ 0.02
93 Ϯ 8
catO
catO
catO
catO
9 Ϯ 1
c
K
K
K
k
(mM)
–
0.30 Ϯ 0.02
93 Ϯ 12
0.32 Ϯ 0.03
mannitol
c
ϩ
(␮M)
–
74 Ϯ 7
1 Ϯ 8
54 Ϯ 3
231 Ϯ 29
775 Ϯ 40
80 Ϯ 10
757
NAD
c
e
f
e
f
ϩ
(␮M)
catO mannitol
–
ND
70 Ϯ 20
ND
3 Ϯ 5
300 Ϯ 50
iNAD
Ϫ1 Ϫ1
c
5
/K
(M
s
)
–
4.9
20
0.015
1400
3.4
20
0.061
2.4
1.0 ϫ 10
Ϫ1 Ϫ1
c
4.0 ϫ 105
2 ϫ 104
k
/K
ϩ
(M
s
)
–
catO NAD
a
a
a,g
Fructose reduction
pH 5.2
pH 5.2
0.8 Ϯ 0.1
19 Ϯ 1
pH 6.8
pH 5.2
8.2 Ϯ 0.1
93 Ϯ 1
pH 7.1
pH 7.1
Ϫ1
Ϫ1
h
k
k
(s
(s
)
0.15 Ϯ 0.01
0.9 Ϯ 0.1
2.7 Ϯ 0.1
93 Ϯ 9
6 Ϯ 1
61 Ϯ 2
catR
obsR
K
K
K
k
c
c
d
)
–
–
403 Ϯ 81
i
(mM)
(␮M)
(␮M)
Ͼ 20
3.9 Ϯ 0.5
22 Ϯ 2
61 Ϯ 10
20 Ϯ 4
41
0.54 Ϯ 0.04
0.24 Ϯ 0.03
80 Ϯ 15
67 Ϯ 4
fructose
iNADH
NADH
e
e
e
ND
ND
ND
64 Ϯ 10
8 Ϯ 3
h
11 Ϯ 2
9 Ϯ 2
16 Ϯ 2
Ϫ1 Ϫ1 i
4
5
/K
(M
s
)
7.4
205
1.5 ϫ 10
450
2.5 ϫ 10
catR fructose
(MϪ1sϪ1)
1.5 ϫ 10
4h
8.9 ϫ 10
4
4.5 ϫ 104
5.1 ϫ 105
3.4 ϫ 105
9.1 ϫ 105
k
/K
catR NADH
a
From fits of Equation 3 to initial-rate data for oxidation and reduction. Equilibrium constants (Keq) for fructose reduction by NADH of 3.4 (N191D), 31 (N300D), and 3.4
(
wild-type enzyme) were calculated from the Haldane relationship K ϭ k
K
K
ϩ
/k
K
K
whereby K
fructose
was not corrected. The kinetically
eq
catR mannitol iNAD
catO fructose iNADH
determined K values are in useful agreement with a K of 9 Ϯ 1 (25 Ϯ 1) determined experimentally at pH 7.1 (6.8) and 25 °C.
eq
eq
b
c
d
e
f
Data from Ref. 13.
Not measurable.
Values of kobs are from Ref. 4.
ND, not determined.
ϩ
ϩ
K
iNAD
is the dissociation constant of enzyme-NAD . However, this does not imply ordered binding, NAD prior to mannitol, in N191D or N300D at pH 7.1. Note that
ϩ
Equation 3 is applicable to both ordered and random kinetic mechanisms.
Data from Ref. 4.
g
h
i
Values were determined at non-saturating concentrations of fructose (2.0 M Ͻ Kfructose).
Values are corrected for the proportion of free carbonyl form (1%) present in aqueous solution of fructose (29).
Steady-state Kinetic Characterization of the Mutants—Ap-
parent kinetic parameters for single-site mutants were obtained
at the optimum pH for mannitol oxidation (pH 10.0) and fruc-
tose reduction (pH 5.2). For the double mutant, kinetic param-
eters in fructose reduction were obtained at pH 5.2. Addition-
ally, a full steady-state kinetic characterization of the wild-type
enzyme and N191D was performed at pH 7.1. Because satura-
tion of N300D with fructose could not be achieved at this pH,
the full kinetic study for N300D was carried out at pH 6.8. All
results are summarized in Table 2. Due to the low activity of
ϩ
N191D and N300D, it was difficult to acquire rates at NAD
concentrations smaller than 60 ␮M. The parameters K
ϩ
iNAD
ϩ
(
(
apparent dissociation constant for enzyme-NAD ) and K
ϩ
NAD
Michaelis-Menten constant) are therefore statistically not well
determined for N191D and N300D, respectively. However, the
internal consistency of each set of kinetic parameters at pH 7.1
FIGURE 3. Free energy profiles for fructose reduction catalyzed by wild-
type enzyme (solid line), N191D (dashed lines), and N300D (dotted line).
(
(
or pH 6.8) was confirmed using Haldane relationship analysis
Table 2) in which the kinetically determined equilibrium con-
⌬G values were calculated as described under supplemental Results, assum-
ing a 1 M standard state for all reactants. E, A, B, P, and Q are enzyme, NADH,
stant is compared with the thermodynamic equilibrium con-
stant of the reaction (20).
ϩ
fructose, mannitol, and NAD , respectively. EA, EAB, EPQ, and EQ are the cor-
TS
responding enzyme complexes. E is the transition state of reaction.
4
N191D and N300D showed large losses (ϳ2 ϫ 10 -fold) in
efficiency, in terms of both k
/K
and k
/K
ϩ
at reasonably explained as an additive effect of the individual site-
catO mannitol
catO NAD
pH 10.0, as compared with wild-type enzyme. These losses directed substitutions. The clear asymmetry of the effects, replace-
reflect almost exclusively the corresponding decreases in k
ment of Asn-300 being ϳ70-fold more strongly disruptive on
/K than is replacement of Asn-191, was not expected
catO
for the mutants in comparison to k
for native PfM2DH. In
k
catO
catR fructose
fructose reduction at pH 5.2, losses in k /K
for N191D from the balanced structural arrangement of active-site groups in
catR fructose
(
17-fold) and N300D (1200-fold) were mainly due to analogous the native enzyme (Fig. 1B). However, considering the uncertainty
decreases in k
as compared with the corresponding param- level associated with current enzyme active-site redesign, the dif-
catR
eters of wild-type PfM2DH. In N300D, apparent binding of ference between N191D and N300D in terms of k /K
was
catR fructose
fructose was weakened 16-fold as compared with native accepted as observed and not further pursued.
enzyme. k /K was relatively insensitive (Յ10-fold Fig. 3 shows free energy profiles for NADH-dependent
change) to substitution of Asn-191 and Asn-300. The observed reduction of fructose catalyzed by wild-type enzyme (pH 7.1),
catR NADH
4
3
.4 ϫ 10 -fold loss in k /K
for the double mutant is N191D (pH 7.1), and N300D (pH 6.8), constructed from the
catR fructose
3
0648 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 40•OCTOBER 1, 2010

From Dehydrogenase to Reductase by Active-site Redesign
corresponding kinetic parameters in Table 2. By comparing the
energy levels for central ternary complexes in reduction (EAB)
and oxidation (EPQ), it is immediately recognized from Fig. 3
that each Asn 3 Asp substitution resulted in very large pertur-
bation of the internal equilibrium of the enzymatic conversion,
as will be discussed later. Another clear consequence of the
mutations is destabilization of the transition state of the reac-
tion. The two mutants can be distinguished according to the
effect of the site-directed substitution on the ratio K
ϩ
/
iNAD
K
ϩ
, which increased ϳ700-fold for N300D but decreased
NAD
1
0-fold for N191D as compared with the corresponding wild-
type ratio of 0.10 at neutral pH (Table 2). The presence of man-
ϩ
nitol thus enhanced the apparent affinity of N191D for NAD ,
ϩ
whereas it had the opposite effect on NAD binding in N300D.
ϩ
Binding of NAD to free enzyme was 27-fold tighter in N191D,
whereas it was unchanged in N300D as compared with wild-
type PfM2DH.
Transient Kinetic Analysis of Reactions Catalyzed by Wild-
type PfM2DH and Mutants Thereof—To examine changes in
the location of the rate-determining step(s) for reactions cata-
lyzed by N191D and N300D as compared with the correspond-
ing reactions catalyzed by the wild-type enzyme that have been
well characterized (4, 13, 18), we performed rapid-mixing,
stopped-flow experiments with these two mutants. Fig. 4 shows
time courses of NADH consumption during reduction of fruc-
tose by N191D and N300D at pH 5.2 (Fig. 4A) and by N191D at
pH 7.1 (Fig. 4B). In each case, the reaction proceeded with a fast
pre-steady-state “burst” that was followed by a linear steady-
state phase. Data were fitted with Equation 4, and relevant tran-
sient rate constants (k
) are summarized in Table 2 along
obsR
with published k
values for the wild-type enzyme. The full
obsR
set of constants is given in supplemental Table S2. k
of
obsR
N191D was not dependent on pH in the range 5.2–7.1. Each
site-directed replacement caused a relatively modest decrease
in k
(N191D: 4.3-fold; N300D: 21-fold) as compared with
obsR
the wild-type reference. In the stopped-flow time courses
shown in Fig. 4A, the amount of NADH (per enzyme equiva-
lent) consumed in the “burst” phase was 4.5-fold higher for
N191D as compared with N300D (see also supplemental Table
S2). This observation appears to be inconsistent with a ratio of
rate constants k
/k
that is higher for N300D than it is for
obsR catR
N191D. However, in terms of K
at the steady state (Table
fructose
2
), N300D binds fructose 7.2-times more weakly than does
N191D. The portion of enzyme tied in the reactive ternary com-
plex under stopped-flow reaction conditions should thus be
smaller for N300D than N191D. Additionally, the relative
amount of productively bound enzyme could be different for
the two mutants.
FIGURE 4. Multiple turnover, stopped-flow progress curves for fructose
Fig. 4C depicts stopped-flow progress curves for mannitol reduction and mannitol oxidation catalyzed by N191D and N300D. A and
B show time courses of fructose reduction at pH 5.2 (I, N191D; II, N300D) and
pH 7.2 (N191D), respectively. C shows time courses of mannitol oxidation
oxidation by N191D and N300D at pH 10.0. After normaliza-
tion for the total concentration of enzyme used ([E]), slopes of
(
I, N191D; II, N300D) at pH 10.0. Results of relevant controls performed under
the linear time courses of NADH formation corresponded to otherwise identical conditions lacking the enzyme are shown in each panel.
Supplemental Table S1 summarizes details of the experiments. The concen-
trations of fructose and mannitol were 2 M and 20 mM, respectively. Fits of
Equation 4 to the data are depicted as gray lines.
k
(Table 2) within limits of the experimental error (Ϯ10%).
catO
The transient rate constant for mannitol oxidation (k
) is
obsO
therefore approximately equal to k
(Table 2). Comparison
catO
between k
for native PfM2DH and k
for N191D and oxidation of mannitol. The large difference in disruptive effect
obsO
catO
N300D reveals that both mutants have lost massively, by ϳ6 of each Asn 3 Asp mutation on rate constants for oxidation
orders of magnitude, their wild-type catalytic ability to promote (k
OCTOBER 1, 2010•VOLUME 285•NUMBER 40
or k
) and reduction (k
) is striking.
obsO
catO
obsR
JOURNAL OF BIOLOGICAL CHEMISTRY 30649

From Dehydrogenase to Reductase by Active-site Redesign
ϩ
enzyme-NAD complexes of N191D and N300D to which the
TABLE 3
Kinetic isotope effects on kinetic parameters
pK value in the respective pH profile of log(k
/K
) can
a
catO mannitol
Parameter
pH
N300D
N191D
Wild-type
be assigned. Catalytic function of the lysine as a Brønsted base
thus appears to have been retained in the mutants. However,
considering the perturbation of active-site electrostatics
expected from an Asn 3 Asp substitution, displacement of the
D
D
D
D
a
k
10.0
10.0
10.0
7.1
1.2 Ϯ 0.1
1.0 Ϯ 0.2
1.0 Ϯ 0.2
1.23 Ϯ 0.03
1.1 Ϯ 0.1
1.2 Ϯ 0.2
1.5 Ϯ 0.3
1.07 Ϯ 0.04
1.9 Ϯ 0.1
2.9 Ϯ 0.2
3.4 Ϯ 0.3
2.0 Ϯ 0.2
2.8 Ϯ 0.2
NDb
1.10 Ϯ 0.04
1.80 Ϯ 0.20
0.94 Ϯ 0.13
catO
catO mannitol
a
a
k
k
k
/K
/K
ϩ
catO NAD
b
c
ND
1.4 Ϯ 0.2
catR
b
5
.2
1.0 Ϯ 0.2
2.2 Ϯ 0.3
2.3 Ϯ 0.3
ND
DkcatR/K
a
pK of Lys-295 from where it was in native PfM2DH (pK ϭ 9.2)
a
a
1
0.0
0.83 Ϯ 0.15
fructose
c
c
8
7
5
.0
.1
.2
2.5 Ϯ 0.1
was surprisingly small in N191D (⌬pK Ϸ 0) and N300D
a
b
ND
2.5 Ϯ 0.1
b
(⌬pK Ϸ ϩ0.8). The absence of a transient lag or a burst phase
a
3.0 Ϯ 0.5
2.1 Ϯ 0.2
NDb
ND
DkobsR
5.2
ND
b
in multiple turnover stopped-flow progress curves of mannitol
7.1
2.0 Ϯ 0.3
oxidation implies that k
of the mutants was limited by ter-
catO
a
b
c
Data are from Ref. 13.
ND, not determined.
Data are from Ref. 18.
D
nary complex conversion. The observation, that KIEs on k
,
catO
k
/K
, and k
/K
ϩ
had similar values, likewise
catO mannitol
catO NAD
supports this notion (for the general case see Ref. 25). Lack of
Primary Deuterium Kinetic Isotope Effects ( KIEs)—We per- pH dependence in k for N191D and N300D therefore sug-
D
catO
formed steady-state and transient kinetic experiments to mea- gests that, as in wild-type enzyme (4), binding of mannitol
D
D
D
sure KIEs on the relevant parameters ( k , k /K
,
causes substantial depression of the pK of Lys-295.
cat
cat substrate
a
D
D
and k
) for N191D and N300D. The analysis of KIEs can
Asp-191 and Asp-300 introduce a pH dependence to k
/
obsR
catR
provide important information on the contribution of the iso-
K
that was not present in the wild-type enzyme (13, 18).
fructose
tope-sensitive step of hydride transfer to rate limitation in the Each Asp arguably represents the ionizable group with appar-
D
D
overall reaction of the mutants. k and k /K
were ent pK of 5.3 Ϯ 0.1 (N191D) and 6.3 Ϯ 0.2 (N300D) that must
cat
cat substrate
a
determined at different pH values, including for each direction be protonated in enzyme-NADH for optimum activity in fruc-
3
of reaction the respective optimum pH as well as a pH value tose reduction (Table 1). Further depression of apparent pK
a
above or below the pK seen in the pH-log(k /K
) profile to a value outside of the measured range (Ͻ5.2) can be assumed
cat substrate
D
(
Table 1). A KIE on k
/K
ϩ
for N191D and N300D was for the double mutant. Retention of activity (in k /K
catO NAD
catR fructose
also determined at pH 10.0. Results are shown in Table 3 along terms) at pH Ն 9 where either Asp of N191D and N300D would
D
with relevant KIEs for the wild-type enzyme that were taken be completely ionized suggests that the pK of Lys-295 in
a
D
from literature. The KIEs on kinetic parameters for mannitol enzyme-NADH was increased in the two mutants to a value
oxidation by N191D and N300D were hardly different from (Ͼ10.0) that was also out of the experimental pH range (Fig. 2B
D
unity (no KIE). In fructose reduction by the two mutants, by and Table 1). It would seem therefore that the negative charge
D
contrast, a substantial KIE of ϳ2 or greater was measured on on the nearby Asp locks Lys-295 in the required protonation
k
/K
across the examined pH range. The correspond- state for reduction. It was shown in previous studies (13, 18)
catR fructose
D
D
D
ing k
was smaller than k /K
. The pH dependence that k /K
for the wild-type enzyme decreases to the
catR
catR fructose
catR fructose
D
D
of k /K
for the mutants was different from that for the equilibrium KIE at high pH due to deprotonation of Lys-295.
catR fructose
D
wild-type enzyme (Table 3).
By contrast,
k
/K
for N191D and N300D did not
catR fructose
Considering the presence of a pre-steady-state burst of display a similar pH dependence in the high pH region (see
NADH consumption in stopped-flow progress curves of fruc- Table 3).
tose reduction by N191D and N300D (Fig. 4), it was also of
The presence of a pre-steady-state burst of NADH consump-
D
interest to obtain a KIE on k
for each mutant. k
is a tion in multiple turnover stopped-flow progress curves of fruc-
obsR
obsR
D
kinetic parameter that includes all steps of the reaction up to tose reduction by the mutants and the pattern of KIEs where
D
D
the one that is rate-limiting in the steady state, and the value of
k
/K
Ͼ k
Ϸ 1 indicates that k
of N191D and
catR fructose
catR
catR
D
k
informs about the role of hydride transfer in being rate- N300D at the optimum pH of 5.2 is limited by release of the
obsR
ϩ
determining for this particular series of steps. We determined second product, which could be mannitol or NAD (see
D
D
k
for N191D and N300D at pH 5.2. A reference value below). The observation, that KIEs on the pre-steady-state
obsR
D
of k
was obtained for wild-type PfM2DH at pH 7.1. Note: rate constant and on k /K
were of comparable magni-
obsR
catR fructose
comparison of reactions catalyzed by native enzyme and tude, is consistent with the proposed kinetic scenario. The
D
mutants thereof using k
values at a single pH was not pos- absence of a pH dependence on k
for the mutated enzymes
obsR
catR
sible because of very small “burst phases” at pH 5.2 (wild-type in the pH range 5.2–7.1 (where substrate binding affinity was
PfM2DH) or 7.1 (mutants; see Fig. 4B). However, the shown sufficiently high to allow determination of the k ) may there-
cat
D
k
values (Table 3) are for near-optimum pH conditions for fore simply reflect the kinetic complexity of this rate parameter.
obsR
fructose reduction by each enzyme. They are similar for all An alternative explanation for the pH independence of k
for
catR
three enzymes studied and are comparable to the correspond- N191D and N300D is that the pK of Asp-191 and Asp-300 is
a
D
ing value of k /K
.
catR fructose
DISCUSSION
3 D
KIEs on kcatR/Kfructose show that hydride transfer was partially rate-limiting
for this rate parameter of N191D and N300D. Therefore, although the pH
dependence of kcatR/Kfructose for the two mutants may still be kinetically
complex, it probably reflects the pH dependence of the chemical reaction
step.
Interpretation of pH-rate Dependences for the Mutants of
PfM2DH—In accordance with previous studies of the wild-type
enzyme (12–14, 18), Lys-295 is the most likely group in
3
0650 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 40•OCTOBER 1, 2010

From Dehydrogenase to Reductase by Active-site Redesign
5
strongly increased upon changing from substrate-limited 1041) in the wild-type enzyme (4) to 1.7 ϫ 10 (ϭ 93/0.00055)
4
5
(
k
/K
; enzyme-NADH) to substrate-saturated (k
)
in N191D and 1.3 ϫ 10 in N300D. The internal equilibrium
catR fructose
catR
reaction conditions. Formation of a hydrogen bond between for wild-type PfM2DH can be compared within the same order
the protonated carboxyl group of Asp and the carbonyl oxygen of magnitude to the thermodynamic equilibrium constant of
of bound fructose could be responsible for this pK change. the reaction (Table 2).
a
However, we must emphasize that ambiguity remains in the
What is the catalytic principle underlying the observed “one-
interpretation of the pH dependence of k
for the two way catalysis” (26) by N191D and N300D as compared with the
catR
D
mutants. The pH dependence of k /K
in the pH range wild-type enzyme that provides effective catalysis to the reac-
catR fructose
5
.2–8.0, where Asp-191 or Asp-300 would undergo deprotona- tion in both directions? Transition state complementarity for
tion while Lys-295 remained protonated, supports a mecha- the native enzyme has been clearly lost as result of either site-
nism in which the ionization state of aspartate affects the iso- directed substitution, as shown in Fig. 3. However, results in
tope-sensitive step of hydride transfer. In summary, therefore, Fig. 3 also reveal that the active site containing Asp-191 or Asp-
pH-profile analysis for the direction of fructose reduction pro- 300 has acquired a substantial amount of complementarity with
vides clear evidence of a new ionizable group in both N191D respect to mannitol, as compared with the wild-type enzyme. A
and N300D, as compared with wild-type enzyme, which maximum amount of binding energy will therefore be realized
appears to contribute to a strongly acidic environment in the when mannitol binds to N191D and N300D while binding of
active sites of the two mutants.
fructose is expected to involve a substantial amount of destabi-
lization energy, affecting substrate, the mutated enzyme or
both. Selective rate acceleration to fructose reduction may be
derived from this destabilization, providing an explanation for
why loss of transition state stabilization in N191D and N300D
as compared with wild-type PfM2DH had almost no effect on
Reaction Coordinate Analysis for N191D and N300D: The
Mutants Behave as Fructose Reductases—Selective disruption
of k
/K
ϩ
as compared with k /K
in N191D and,
catO NAD
catR NADH
to a lesser extent, in N300D (Table 2) is difficult to reconcile
with an Ordered Bi Bi kinetic mechanism where these catalytic
efficiencies stand for coenzyme binding to free enzyme. How-
ever, in accordance with previous studies of PfM2DH showing
that individual substitutions of Asn-191 and Asn-300 by Ala
decreasing k
. Stabilization of mannitol is expected to
obsR
involve productive as well as nonproductive modes of binding.
Evidence from MD simulation clearly supports non-productive
binding of mannitol whereby two different orientations of the
reactive C2-OH of the substrate appear to be preferred, as
shown in Scheme 1A and, in more detail, in supplemental Fig.
S4. One set of conformations frequently sampled in the simu-
lation of either mutant involved a bidentate hydrogen bond
from Asp-230 to C1 and C2 hydroxy groups of mannitol. This
conformation was especially predominant in N300D. The other
conformational ensemble featured a hydrogen bond between
the C2-OH and Asp, especially Asp-191, whereas the C1-OH
remained bonded to Asp-230. Scheme 1A shows representative
structure snapshots from the MD simulation. Full time courses
of simulated atom-atom distances are given in supplemental
ϩ
caused loss of order in binding of NAD and mannitol (4), we
believe that, under conditions of a saturating mannitol concen-
tration, k
/K
ϩ
for N191D and N300D is really the sec-
catO NAD
ond-order rate constant for reaction of enzyme-mannitol with
ϩ 4
NAD . These details of the kinetic mechanism of N191D and
N300D are, however, not essential for the conclusions drawn
herein and were therefore not further pursued. Notwithstand-
ing, Fig. 3 might show only one of two possible routes of forma-
ϩ
tion of enzyme-NAD -mannitol in the two mutants.
ϩ
Mutation of Asn-191 stabilized enzyme-NAD -mannitol by
Ϫ1
Ϫ20.6 kJ mol , whereas it destabilized enzyme-NADH-fruc-
Ϫ1
tose by ϩ11.5 kJ mol , relative to the corresponding com-
plexes of wild-type PfM2DH. The kinetic barrier for mannitol
Fig. S4 (A–C). The experimental observation that k
was
catO
Ϫ1
oxidation was strongly increased (⌬⌬G ϭ ϩ35.8 kJ mol ) as a
3
ϳ10 -fold more sensitive than k
/K
to mutation of
catO mannitol
result of the mutation, whereas it was hardly affected for fruc-
Asn-191 and Asn-300 is likewise consistent with nonproduc-
tive binding of mannitol (Scheme 1A), implying that a substan-
tial fraction of each mutant will be tied in a ternary complex that
is unable to yield product and can regenerate the active enzyme
only by dissociation. In the productive complex at the optimum
pH for mannitol oxidation (Scheme 1B), the negatively charged
side chain of Asp-191 or Asp-300 might be oriented away from
Ϫ1
tose reduction (⌬⌬G ϭ ϩ3.5 kJ mol ). Loss of “transition
state” stabilization energy in N191D as compared with wild-
Ϫ1
type enzyme was ϳ15.2 kJ mol . The overall pattern of
changes in free energy profile resulting from site-directed sub-
stitution of Asn-300 by Asp was quite similar to the one just
described for N191D. Differential stabilization of ternary com-
plexes undergoing oxidation and reduction, as shown for the
two mutants in Fig. 3, results in a dramatic change of internal
Lys-295. The proposed conformer provides a tentative expla-
ϩ
nation for why the pK of Lys-295 in enzyme-NAD is rela-
a
equilibrium (K ϭ k
/k
), from a value of 0.4 (ϭ 403/
int
obsR obsO
tively insensitive to site-directed substitution Asn 3 Asp.
Nonetheless, the catalytically important “activation” of the
reactive alcohol group in substrate through partial proton
abstraction by Lys-295 (Scheme 1C) is expected to be disfa-
4
Kinetic data in Table 2 show that binding of mannitol enhances the affinity
ϩ
of N300D for NAD (KiNAD
ϩ
ӷ KNAD
ϩ
). By contrast, the affinity of N191D for
Ӷ KNAD ). In the crystal
ϩ
NAD is weakened by binding of mannitol (KiNAD
ϩ
ϩ
ϩ
structure of wild-type PfM2DH bound with NAD (PDB: 1LJ8), Asn-191 vored electrostatically in both N191D and N300D.
points towards the nicotinamide ring of coenzyme. We therefore believe
ϩ
that ϳ30-fold tighter binding of NAD by free N191D as compared to
ϩ
NAD binding by wild-type enzyme and N300D reflects attraction
5
between the negatively charged Asp and the positively charged nicotina-
mide moiety. From the crystal structure, similarly strong interactions are
not expected for Asp-300.
The value of Kint for N300D (at pH 6.8) was calculated using the thermody-
namic relationship, ⌬⌬G ϭ ⌬GEPQ Ϫ ⌬GEAB ϭ ϪRT ln Kint). ⌬GEPQ (ϭ Ϫ38
kJ/mol) and ⌬GEAB (ϭ Ϫ14.6 kJ/mol) are from Fig. 3.
OCTOBER 1, 2010•VOLUME 285•NUMBER 40
JOURNAL OF BIOLOGICAL CHEMISTRY 30651

From Dehydrogenase to Reductase by Active-site Redesign
Proposed Catalytic Mechanism of
N191D and N300D—The proposed
catalytic function of Lys-295 and
Asp-191 (or Asp-300) in facilitating
reduction of fructose by NADH is
shown in Scheme 2. Herein, the
active site of the mutated mannitol
dehydrogenases provides a “super-
acidic” micro-environment, aiding
in a very strong polarization of the
substrate carbonyl, which in turn is
expected to promote the hydride
transfer from NADH. Lys-295
retains its native role (14) as cata-
lytic Brønsted acid in N191D and
N300D. The proposed arrangement
of catalytic groups as well as their
function in carbonyl group polariza-
4
tion have strong analogy to ⌬ -3-ke-
tosteroid 5␤-reductase from the
aldo-keto-reductase protein super-
family (15, 27). The 5␤-reductase
catalyzes the double bond reduction
4
of the ⌬ -ene by NADPH and uses
a diad of glutamic acid and tyro-
sine for interaction with the sub-
5
strate carbonyl. ⌬ -3-Ketosteroid
isomerase catalyzes isomeriza-
5
tion of 3-oxo-⌬ -steroids to their
4
⌬
-conjugated isomers. Using as-
partic acid and tyrosine, this
enzyme shows a similarly “super-
acidic” binding pocket for the
3
-keto group of the substrate as the
␤-reductase (28).
5
In conclusion, redesign of the
oxyanion hole of PfM2DH through
substitution of Asn-191 or Asn-300
by Asp brought about a drastic
change in the directional preference
of enzyme catalysis. A double
mutant harboring both Asn 3 Asp
substitutions was devoid of measur-
able activity in mannitol oxidation
but performed as weakly active fruc-
tose reductase under low pH condi-
tions. This is to our knowledge the
first time that a parent dehydrogen-
ase was converted into a reductase
through targeted modification of
the catalytic center. Optimal cataly-
sis of the reaction in both directions
SCHEME1. Proposedbindingmodesformannitol(A,nonproductive;B,productive)inN191DandN300D is a feature of the wild-type enzyme,
as compared with mannitol binding in wild-type enzyme (C). A shows representative structure snapshots
which seems to reflect complemen-
for an MD simulation of the ternary enzyme-NADH-mannitol complex of N191D (left) and N300D (right). The
unnumbered Asp shown in A and B may be either Asp-191 or Asp-300. For clarity, only the reactive groups of tarity of the active site to the transi-
ϩ
mannitol and NAD are displayed. C shows the proposed (partial) abstraction of the substrate proton in the
ternary complex prior to undergoing hydride transfer to NAD . A structure snapshot from the MD simulation
is also shown.
tion state. By strongly stabilizing
ϩ
bound mannitol in productive as
well as non-productive conformers,
3
0652 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 40•OCTOBER 1, 2010

From Dehydrogenase to Reductase by Active-site Redesign
4
. Klimacek, M., and Nidetzky, B. (2010) Biochem. J. 425, 455–463
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8
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(
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. Eschenburg, S., Genov, N., Peters, K., Fittkau, S., Stoeva, S., Wilson, K. S.,
and Betzel, C. (1998) Eur. J. Biochem. 257, 309–318
1
1
0. Bobofchak, K. M., Pineda, A. O., Mathews, F. S., and Di Cera, E. (2005)
J. Biol. Chem. 280, 25644–25650
1. Kavanagh, K. L., Klimacek, M., Nidetzky, B., and Wilson, D. K. (2002)
J. Biol. Chem. 277, 43433–43442
1
1
1
2. Klimacek, M., and Nidetzky, B. (2002) Biochem. J. 367, 13–18
3. Klimacek, M., and Nidetzky, B. (2002) Biochemistry 41, 10158–10165
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1
5. Di Costanzo, L., Drury, J. E., Christianson, D. W., and Penning, T. M.
(2009) Mol. Cell. Endocrinol. 301, 191–198
SCHEME 2. Proposed catalytic mechanism of fructose reduction by
super-acidic” catalytic centers of N191D and N300D. Only the reactive
groups of the substrate and NAD(H) are shown.
“
16. Di Costanzo, L., Penning, T. M., and Christianson, D. W. (2009) Chem.
Biol. Interact. 178, 127–133
N191D and N300D by contrast achieve essentially one-way
catalysis of fructose reduction, characterized by a high maximal
1
7. Di Costanzo, L., Drury, J. E., Penning, T. M., and Christianson, D. W.
(2008) J. Biol. Chem. 283, 16830–16839
rate (not very different from that of the wild-type enzyme in 18. Slatner, M., Nidetzky, B., and Kulbe, K. D. (1999) Biochemistry 38,
1
0489–10498
k
as well as k
terms) accompanied by weaker binding of
obsR
catR
1
9. Northrop, D. B. (1977) in Isotope Effects on Enzyme-catalyzed Reactions
the ketose substrate. Design of “super-acidic” catalytic centers
capable of strong polarization of the reactive carbonyl group
could be of general relevance in the development of essentially
uni-directional reductases to be applied as biocatalysts for
organic synthesis. These engineered reductases are expected
(
Cleland, W. W., O’Leary, M. H., and Northrop, D. B., eds) University Park
Press, Baltimore, MD
2
0. Segel, I. H. (1993) Enzyme Kinetics: Behavior and Analysis of Rapid Equi-
librium and Steady-state Enzyme Systems, John Wiley & Sons Inc., New
York
to show optimum activity at low pH where as an additional 21. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel,
R. D., and Bairoch, A. (2005) in The Proteomics Protocols Handbook
advantage, oxidation of alcohol product is disfavored
(
Walker, J. M., ed) Humana Press Inc., Totowa, NJ
thermodynamically.
2
2
2
2. Hess, B., Kutzner, C., Van Der Spoel, D., and Lindahl, E. (2008) J. Chem.
Theory Comput. 4, 435–447
Acknowledgments—We are grateful to Dr. Rene Meier for MD simu-
lations and thank Veronika Milocco, Valentin Pacher, and Julia
Hartl for expert technical assistance. Prof. Walter Keller (Institute of
Molecular Biosciences, University of Graz, Austria) is thanked for
assistance during recording CD spectra.
3. Oostenbrink, C., Villa, A., Mark, A. E., and van Gunsteren, W. F. (2004)
J. Comput. Chem. 25, 1656–1676
4. Humphrey, W., Dalke, A., and Schulten, K. (1996) J. Mol. Graph. 14,
3
3–38, 27–38
25. Cook, P. F. (1991) in Enzyme Mechanism from Isotope Effects (Cook, P. F.,
ed) CRC Press, Boca Raton, FL
2
2
6. Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219–410
7. Faucher, F., Cantin, L., Luu-The, V., Labrie, F., and Breton, R. (2008)
Biochemistry 47, 8261–8270
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JOURNAL OF BIOLOGICAL CHEMISTRY 30653