(Received for publication, May 30, 1996, and in revised form, October 7, 1996)
From the Biochemistry Laboratory, Gifu Pharmaceutical
University, Gifu 502, Japan and the ¶ Department of
Bioengineering, Nagaoka University of Technology, Nagaoka,
Niigata 940-21, Japan
Mouse lung carbonyl reductase, a member of the
short-chain dehydrogenase/reductase (SDR) family, exhibits coenzyme
specificity for NADP(H) over NAD(H). Crystal structure of the
enzyme-NADPH complex shows that Thr-38 interacts with the 2-phosphate
of NADPH and occupies the position spatially similar to an Asp residue of the NAD(H)-dependent SDRs that hydrogen-bonds to the
hydroxyl groups of the adenine ribose of the coenzymes. Using
site-directed mutagenesis, we constructed a mutant mouse lung carbonyl
reductase in which Thr-38 was replaced by Asp (T38D), and we compared
kinetic properties of the mutant and wild-type enzymes in both forward and reverse reactions. The mutation resulted in increases of more than
200-fold in the Km values for NADP(H) and decreases of more than 7-fold in those for NAD(H), but few changes in the Km values for substrates or in the
kcat values of the reactions. NAD(H) provided
maximal protection against thermal and urea denaturation of T38D, in
contrast to the effective protection by NADP(H) for the wild-type
enzyme. Thus, the single mutation converted the coenzyme specificity
from NADP(H) to NAD(H). Calculation of free energy changes showed that
the 2
-phosphate of NADP(H) contributes to its interaction with the
wild-type enzyme. Changing Thr-38 to Asp destabilized the binding
energies of NADP(H) by 3.9-4.5 kcal/mol and stabilized those of NAD(H)
by 1.2-1.4 kcal/mol. These results indicate a significant role of
Thr-38 in NADP(H) binding for the mouse lung enzyme and provide further
evidence for the key role of Asp at this position in NAD(H) specificity of the SDR family proteins.
In several animal lungs (1-5), carbonyl reductase (NADPH) (CR1; EC 1.1.1.184) catalyzes the reduction of various aliphatic and aromatic carbonyl compounds and the oxidation of secondary alcohols and aliphatic aldehydes. The enzyme is highly expressed in bronchiolar and alveolar epithelial cells (3, 6) and has been thought to function in pulmonary metabolism of endogenous carbonyl compounds such as aliphatic aldehydes and ketones derived from lipid peroxidation, 3-ketosteroids, and fatty aldehydes, as well as in xenobiotic metabolism.
The cDNAs for pulmonary CRs of mouse (7) and pig (8) have been
cloned, and their deduced amino acid sequences (composed of 244 residues with 85% identity between them) indicate that the enzymes
belong to the short-chain dehydrogenase/reductase (SDR) family, which
includes a large number of prokaryotic and eukaryotic enzymes with
different specificities for coenzymes and substrates (9, 10). The
pulmonary CR sequences contain two consensus sequences of
Tyr-X-X-X-Lys and
Gly-X-X-X-Gly-X-Gly that
are demonstrated to be the active site and coenzyme binding domains,
respectively, by site-directed mutagenesis (Ref. 9 and references
therein) and x-ray crystallography studies (11-14) of several SDR
family proteins. The region around the latter sequence of the SDRs
forms a fold that is characteristic of the coenzyme-binding fold in dehydrogenases of other families (15, 16). The NAD(H)- and
NADP(H)-dependent dehydrogenases of other families have
different fingerprint sequences for coenzyme binding. The
NAD(H)-dependent enzymes have an invariant
Gly-X-Gly-X-X-Gly sequence and an
acidic residue (usually Asp) at the C terminus of the second
strand, whereas another consensus sequence has been proposed for the
NADP(H)-dependent enzymes in which the third Gly of the
NAD(H)-binding fingerprint is replaced by Ala, and a positively charged
residue is usually included in the neighborhood of the C terminus of
the
fold (15-20). For the SDR family, both NAD(H)- and
NADP(H)-dependent enzymes possess the same
Gly-X-X-X-Gly-X-Gly
pattern. Although a factor determining the specificity for NAD(H) has
been proposed to be Asp at the C terminus of the second
strand of
the coenzyme-binding fold (9-14), the residue(s) responsible for
NADP(H) specificity remain unknown.
We have recently solved the three-dimensional structure of mouse lung
CR, which exhibits high coenzyme preference for NADP(H) over NAD(H),
and have shown that Lys-17 and Arg-39, which exist before the second
Gly of the Gly-rich pattern and at the C terminus of the
fold, respectively, are responsible for the coenzyme specificity (21).
The roles of the two basic amino acids have been confirmed by their
site-directed mutagenesis (22). In addition, comparison of crystal
structures between mouse lung CR and the NAD(H)-dependent
SDRs has suggested that, while the Asp residue at the coenzyme-binding
fold forms a bifurcated hydrogen bond to the adenine ribose for the
NAD(H)-dependent enzymes (11-14), Thr-38 of CR at a
position corresponding to the Asp residue hydrogen-bonds to the
2
-phosphate of NADPH through a water molecule (21).
In this study, we used site-directed mutagenesis to replace Thr-38 with Asp (T38D) and compared the kinetic and thermodynamic properties of coenzyme binding to the wild-type CR and T38D. The results show that the single mutation converts the coenzyme specificity of mouse lung CR from NADP(H) to NAD(H).
Pyridine nucleotide coenzymes and pI markers were obtained from Oriental Yeast (Tokyo, Japan); restriction and DNA-modifying enzymes were from Nippon Gene (Tokyo, Japan) and Takara Shuzou (Osaka, Japan); Escherichia coli cells and plasmids were from Stratagene. The cDNA for mouse lung CR and the antibody against the enzyme previously prepared (7) were used. The oligonucleotide primers for the site-directed mutagenesis were synthesized as described (7, 22). All other chemicals were of the highest grade that could be obtained commercially.
Site-directed Mutagenesis, Expression, and PurificationThe
cDNA for T38D was generated using a modified overlap-extension
technique (23) as described previously (22), using the partially
complementary primer pairs (forward
(5-GTGGCGGTG
CGGACCAA-3
) and reverse
(5
-TTGGTCCG
CACCGCCAC-3
)) that contained a codon of an
altered amino acid (underlined). The complete coding region of the
mutated cDNA was sequenced as described previously (8) to confirm
the presence of the desired mutation and to ensure that no other
mutation had occurred.
The wild-type and mutated cDNAs were expressed in E. coli (JM105) cells, and the recombinant enzymes were purified from the 12,000 × g supernatants of the homogenates of the cells (each a 1-liter culture) as described previously (7).
Characterization of Protein SamplesProtein concentration of the crude extract and enzyme preparations during the purification was determined by the Bradford method (24) using bovine serum albumin as the standard. The molecular mass of the CR subunit was determined by SDS-polyacrylamide gel electrophoresis (25) on 12.5% gels. The pI value of the purified enzyme was assessed by isoelectric focusing (26) on 7.5% polyacrylamide gels containing 2% Ampholite (Pharmacia Biotech Inc.) and 8 M urea using the pI markers. Western blot immunoanalysis using the antibody against CR was carried out as described previously (7).
Enzyme Assay and Kinetic AnalysisReductase and dehydrogenase activities of CR were assayed by recording the rate of change in NAD(P)H absorbance at 340 nm, except that an absorbance at 366 nm was monitored in the assay with high concentrations of NAD(P)H. The standard reaction mixture for the dehydrogenase activity consisted of 80 mM potassium phosphate buffer, pH 7.0, 0.5 mM NAD(P)+, 2.0 mM cyclohex-2-en-1-ol (CHX), and enzyme in a total volume of 2.0 ml. The reductase activity was determined with NAD(P)H and pyridine-3-aldehyde (P3A) as the coenzyme and substrate, respectively. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation and oxidation of 1 µmol of NAD(P)H/min at 25 °C.
The kinetic mechanism and constants of the P3A reduction were analyzed according to the method of Cleland (27). The initial velocities were fitted to the equation
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(Eq. 1) |
For thermal inactivation, the enzymes (0.1 mg/ml) were incubated at 34 °C in 0.1 M potassium phosphate buffer, pH 7.0, containing 0.15 M KCl and 0.1% bovine serum albumin in the presence or absence of NAD(P)(H) or substrate. At different times, aliquots of 50 µl from each sample were taken and assayed for the dehydrogenase activity. For the denaturation by urea, the enzyme (30 µg/ml) was incubated at 25 °C for 2 h in 0.1 M Tris-HCl buffer, pH 8.0, containing 0-5 M urea in the presence or absence of NAD(P)+ or substrate. The dehydrogenase activity was expressed as a percentage of that in the absence of urea. This assay is unaffected by the presence of up to 0.1 M urea.
Computer ModelingThe subunit 1 (protein and NADP(H)) in the crystal structure of WT (21) was chosen to build the mutated model structure T38D. The Thr-38 of the subunit was replaced with an Asp residue using the program QUANTA (Molecular Simulations, Burlington, MA). This single-subunit model with the coenzyme NADP(H) was refined through the energy minimization routine incorporated in the program X-PLOR (29).
When
the CHX dehydrogenase activity in the lysate of the E. coli
cells was assayed with NADP+ and NAD+ as the
coenzymes, the mutant enzyme T38D showed pronounced preference for
NAD+. The ratio of the NAD+-linked to the
NADP+-linked activities was 18, which was much higher than
the value of 0.1 for WT. The mutant enzyme was purified to apparent
homogeneity on SDS-polyacrylamide gel electrophoresis (Fig.
1A) and isoelectric focusing (Fig.
1B). The NADP+- and NAD+-linked
activities of the purified T38D were 0.39 unit/mg and 6.6 units/mg,
respectively, whereas the respective values of the purified WT were 4.5 units/mg and 0.70 unit/mg. Although the T38D and WT showed the
identical subunit molecular mass and reactivity with the CR antibody on
Western blot immunoanalysis (data not shown), the pI value of 8.8 for
T38D was lower than the pI value of 9.3 for WT due to the introduction
of an acidic charge of Asp.
Kinetic Alteration by Mutagenesis
The kinetic effect of the mutation was assessed by comparing the kinetic parameters in both forward (with P3A as the substrate) and reverse (with CHX as the substrate) directions between WT and T38D (Table I). In the forward reaction, the most striking alteration by the mutagenesis of T38D was to increase the Km and KIA for NADPH in the NADPH-linked reaction and to decrease the Km and KIA for NADH in the NADH-linked reaction. The ratio of the kcat/Km for the NADPH-linked to the NADH-linked reactions indicates an approximately 1,300-fold change in coenzyme specificity from one that prefers NADPH to one that favors NADH. In the reverse reaction, similar conversion of the coenzyme preference by the mutation was observed, although all the kinetic constants were apparent values with a fixed concentration of coenzyme or substrate. Thus, the single mutation converted the coenzyme specificity of mouse lung CR from NADP(H) to NAD(H).
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The double reciprocal plots of initial velocity versus
NAD(P)H concentrations at five fixed levels of P3A yielded a series of
intersecting lines for T38D (data not shown). In the analysis of
product inhibition at the saturated concentration of the fixed substrate, noncompetitive inhibition patterns were obtained with NAD+ (Kis = 0.83 mM;
Kii = 1.5 mM) and pyridine-3-methanol (Kis = 31 mM; Kii = 90 mM) when P3A was the variable substrate. The patterns
found with NAD+ and pyridine-3-methanol were competitive
(Kis = 0.19 mM) and uncompetitive
(Kii = 75 mM), respectively, with respect to NADH. In addition to NAD+, its analogs
NADP+, 2-AMP, and 5
-AMP inhibited both WT and T38D
competitively with respect to NAD(P)H (Table II). From
these data, the kinetic mechanism for T38D appears most likely to be an
Ordered Bi Bi mechanism in which the coenzyme binds first to the enzyme
followed by the aldehyde substrate. The product inhibition patterns
with T38D are consistent with those of WT (22) and guinea pig lung CR,
for which a di-iso Ordered Bi Bi mechanism with coenzyme-induced isomerization has been suggested (4). The conformational change upon
the binding NADP(H) was also observed in a computer modeling study2 of mouse lung CR structure with or
without the coenzymes. Since the change of binding to coenzyme will
affect the binding of the substrate in this kinetic mechanism, the
small changes in Km for P3A and CHX (Table I) may be
due to the altered binding of NADP(H) and NAD(H). In addition, the
Kis values for the competitive inhibitors with
respect to the coenzyme imply their dissociation constants in this
kinetic mechanism, and thus the effects of the mutation on the
affinities for the oxidized coenzymes and their analogs can be assessed
by comparing their Kis values between WT and T38D.
The mutation decreased the affinities for NADP+ and 2
-AMP
and increased the affinities for NAD+ and 5
-AMP, although
the changes in the affinities for the AMPs were low because of the
absence of other parts of NAD(P)+ molecules that interacted
with several residues of the enzyme (21). The results indicate that the
adenine ribose moiety of the coenzymes interacts with Thr-38 and the
replaced Asp.
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The kinetic results were also supported by differences in protection
against the thermal and urea denaturation by coenzymes between T38D and
WT. The thermal inactivation of WT was protected completely by low
concentrations of NADP(H) and moderately by high concentrations of
NAD(H), whereas NAD(H) provided more efficient protection against the
thermal inactivation of T38D than did NADP(H) (Fig. 2).
Similar protective effects of the coenzymes appeared on the
denaturation by urea, in which T38D was slightly unstable compared with
WT (Fig. 3). NADP+ showed greater protection
against the urea denaturation of WT than NAD+, whereas
obvious protection of T38D from the denaturation was observed only by
the addition of NAD+. It should be noted that the substrate
(2 mM P3A or CHX) did not show significant protection
against the thermal and urea denaturation of the two enzymes, which
supports the kinetic ordered addition of the coenzymes to the free
enzymes followed by the substrates.
In addition to the loss of the hydrogen bridge between Thr-38 and the
2-phosphate of NADP(H) by the mutation, other structural factors must
be considered to explain the large alteration in the kinetic constants
for the coenzymes. In the crystal structure of the mouse lung
CR·NADPH complex (21), the 2
-phosphate of NADPH has been shown to
interact with the side chains of Lys-17, Thr-38, and Arg-39, of which
Thr-38 occupies the position spatially similar to the Asp of the
NAD(H)-dependent SDRs that interacts with the adenine
ribose of NAD(H) (11-14). In the mutated model structure (T38D), the
distances between each oxygen atom of Asp-38 carboxylate and the
nearest oxygen atom of the NADP(H) 2
-phosphate were 2.8 Å and 3.8 Å.
The calculated electrostatic energy between the Asp-38 and the
2
-phosphate moiety (T38D) was 22 kcal/mol higher than that between the
Thr-38 and the 2
-phosphate moiety (WT). Thus, one of the possible
structural factors for the switch of the coenzyme specificity by the
mutation is an electrostatic repulsion between the negatively charged
2
-phosphate of NADP(H) and the carboxylate group of the replaced Asp.
Another structural factor may be steric hindrance of the side chain of
the replaced Asp residue against the NADP(H) binding. The presence of
the Asp side chain that is larger than that of Thr-38 may interfere
with the proximity of the side chains of Lys-17 and Arg-39 to
2
-phosphate of NADP(H) as suggested (21). Furthermore, the increase in
the affinity for NAD(H) by the mutation suggests that the side chain of
the replaced Asp makes a hydrogen-bonded interaction with the hydroxyl
groups of adenine ribose of the coenzymes as shown in the crystal
structures of the NAD(H)-dependent SDRs (11-14).
Thermodynamic effects of removing
the 2-phosphate of NADP(H) or replacing Thr-38 with Asp on the
coenzyme binding energy were calculated according to the relationships
described by Sem and Kasper (30) (Table III). The change
in the binding energy for 2
-phosphate removal of NADP(H) in the
presence of Thr-38 (i.e. WT) indicates that the 2
-phosphate
groups of NADPH and NADP+ contribute 3.2 and 4.5 kcal/mol,
respectively, of binding energy in their interactions with the enzyme.
The values for 2
-phosphate removal of NADPH and NADP+ for
the mutant T38D were
1.9 and
1.3 kcal/mol, respectively. The
negative values imply that the mutation not only destabilizes the
NADP(H) binding but also stabilizes the NAD(H) binding. When Thr-38 was
replaced with Asp, the binding energies of NADPH and NADP+
were destabilized by 3.9 and 4.5 kcal/mol, respectively, but those of
NADH and NAD+ were conversely stabilized by 1.2 and 1.4 kcal/mol, respectively. Since the values of 3.9 and 4.5 kcal/mol for
the hydrogen bridge made by Thr-38 of mouse lung CR are comparable to
the values for the Ser-2
-phosphate interaction in cinnamyl-alcohol
dehydrogenase (31) and for other salt bridges between NADP(H) and Lys
or Arg reported for mouse lung CR (22) and other enzymes (30-33),
Thr-38 may contribute significantly to the binding energy of NADPH by making a hydrogen bridge to the 2
-phosphate of NADPH. In addition, the
stabilization of the NAD(H) binding energies by the mutation supports
the hydrogen-bonded interaction of the side chain of the replaced Asp
with the hydroxyl groups of adenine ribose of the coenzymes.
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Thr-38 is conserved only in mouse and pig lung CRs (7, 8) of the
NADP(H)-dependent SDR enzymes, several of which have Ser or
Cys (34-36) and most of which have Ala at this position (9, 21).
Although the amino acids with a hydroxyl or sulfhydryl group can be
anticipated to participate in NADP(H) binding in several SDRs, the role
of Ala remains unknown. However, the presence of an Ala residue at this
position in the other SDRs has been suggested to be important for
making the coenzyme-binding cleft to avoid the steric hindrance against
the interactions between the 2-phosphate of NADP(H) and the side
chains of the basic residues corresponding to Lys-17 and Arg-39 of
mouse lung CR (21). That is, since the interactions of the two basic
residues with the 2
-phosphate of the coenzymes in the SDRs containing
Ala at this position may be stronger than those in mouse lung CR, the
additional interaction by Thr would no longer be required for the
coenzyme specificity.
Such a drastic change in coenzyme
specificity by single mutation observed in the present study has not
been reported yet for dehydrogenases of the SDR (37-39) and other
families (17, 19, 40, 41) in which systematic replacement of a set of
amino acids in their folds is necessary to switch their
coenzyme specificity. Although there has been no report on the mutation of the residue corresponding to Thr-38 of mouse lung CR in
NADP(H)-dependent enzymes of the SDR family, the
reverse-sense mutation, i.e. the replacement of the Asp with
other residues, has been described for two NAD(H)-dependent
enzymes of this family. The mutations of Asp-37 to Ile for rat
dihydropteridine reductase (37) and of Asp-39 to Asn for
Drosophila alcohol dehydrogenase (38) increase the
affinities for NADP(H) but do not have a significant effect on those
for NAD(H); therefore, the mutant enzymes still show coenzyme
preference for NAD(H) over NADP(H).
The present study reveals not only the important role of Thr-38 in the
NADP(H) binding to mouse lung CR, but also the key role of Asp at the C
terminus of the second strand of the
fold in the NAD(H)
specificity of the SDR family proteins. Since (unlike dehydrogenases of
other families (15-20)) the SDR family proteins have the same
Gly-X-X-X-Gly-X-Gly
sequence in their
folds (9-14), the most critical
determinant for coenzyme specificity is the presence of Asp at this
position. As T38D, which has Lys-17 and Arg-39 responsible for the
NADP(H) binding (21, 22), showed the coenzyme preference for NAD(H)
over NADP(H), some SDRs with both the Asp and one of the basic residues
corresponding to Lys-17 and/or Arg-39 of mouse lung CR are
NAD(H)-specific (42-44). Conversely, the structural determinant for
the NADP(H) specificity in the SDR family proteins is the replacement
of the Asp with neutral amino acids with shorter side chains in
addition to the presence of the two basic residue(s), as suggested by
comparison of crystal structures of mouse lung CR and several
NAD(H)-dependent SDRs (21).