(Received for publication, August 24, 1995; and in revised form, November 21, 1995)
From the
Nine single genetic mutants of rat dihydropteridine reductase
(EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A,
and A133S and one double mutant, Y146F/K150Q, have been engineered,
overexpressed in Escherichia coli and their proteins purified.
Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been
crystallized and structurally characterized. Kinetic constants for each
of the mutant enzyme forms, except N186A, which was too unstable to
isolate in a homogeneous form, have been derived and in the five
instances where structures are available the altered activities have
been interpreted by correlation with these structures. It is readily
apparent that specific interactions of the apoenzyme with the cofactor,
NADH, are vital to the integrity of the total protein tertiary
structure and that the generation of the active site requires bound
cofactor in addition to a suitably placed W86. Thus when the three
major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is
formed. It is also apparent that tyrosine 146 is vital to the activity
of the enzyme, as the Y146F mutant is almost inactive having only 1.1%
of wild-type activity. However, when an additional mutation, K150Q, is
made, the rearrangement of water molecules in the vicinity of
Lys is accompanied by the recovery of 50% of the
wild-type activity. It is suggested that the involvement of a water
molecule compensates for the loss of the tyrosyl hydroxyl group. The
difference between tyrosine and histidine groups at 146 is seen in the
comparably unfavorable geometry of hydrogen bonds exhibited by the
latter to the substrate, reducing the activity to 15% of the wild type.
The mutant A133S shows little alteration in activity; however, its
hydroxyl substituent contributes to the active site by providing a
possible additional proton sink. This is of little value to
dihydropteridine reductase but may be significant in the sequentially
analogous short chain dehydrogenases/reductases, where a serine is the
amino acid of choice for this position.
The regeneration of the tetrahydrobiopterin cofactor that
participates in the initial hydroxylation reactions of the aromatic
amino acids, phenylalanine, tryptophan, and tyrosine, essential for the
generation of the
catecholamines(1, 2, 3, 4) , is
carried out by the NADH-requiring enzyme, dihydropteridine reductase
(EC 1.6.99.7). Recent reports from this laboratory have described the
crystal structures of this enzyme derived from both rat and human
sources(5, 6) , have identified its classification
into the larger protein families of the short chain dehydrogenases (7) and epimerases(8, 9) , and have suggested
that the Tyr-(Xaa)-Lys-containing motif is essential to the
reductive mechanism of the protein(10) .
During the course
of this investigation, it has become apparent that certain amino acids
are essential to the binding of the dinucleotide cofactor. Of
particular importance are Asp, Lys
, and
Asn
, as each of these amino acids display strong hydrogen
bonds to the adenine ribose, nicotinamide ribose, and nicotinamide
amide substituent, respectively. In addition, Tyr
interacts with both Lys
and the 4-position of the
quinonoid dihydrobiopterin substrate placed by graphic computations at
the ternary complex active site. Moreover, Trp
appears to
orient the plane of the pteridine by steric interactions. It is also
apparent that when a sequence comparison is made between
dihydropteridine reductase and the family of short chain
dehydrogenases/reductases, in the majority of cases Ala
is converted to Ser(11) . We describe here the altered
properties of dihydropteridine reductase when Lys
is
replaced with Gln, Ile, and Met, when Tyr
is replaced by
Phe and His, when a double mutant Tyr
Phe/Lys
Glu is generated, when Trp
is
converted to Ile and when Asn
is converted to alanine. In
addition an Ala
Ser variant of dihydropteridine
reductase has been created to help discover if the prevalence for this
change in the dehydrogenases can be interpreted. Crystal structures of
several of the mutants have been obtained and structural changes
occurring in these derivatives have been used to interpret the altered
kinetic properties of the mutated enzymes. Together, these results
support the concept that the dihydropteridine reductase protein
requires a tightly bound reduced dinucleotide for structural and
mechanistic integrity and that the Tyr-(Xaa)
-Lys motif is
important to the proton transfer that may initiate or complete
substrate reduction.
Examination of the binary complex of dihydropteridine
reductase and NADH (5) indicates that the principal hydrogen
bonding protein interactions occur between the dinucleotide and
Asp, Lys
, and Asn
(Fig. 1). In order to estimate the importance of each of
these sites, they have been mutated to afford differing proteins
containing Asp
Ile(27) , Lys
Gln, Lys
Ile, Lys
Met, and Asn
Ala amino acid changes.
If the ternary complex employing a graphically introduced quinonoid
dihydrobiopterin substrate is also analyzed(5) , a further site
of interaction appears between substrate and Tyr
.
Mutations of this site to Phe or His have also been carried out and
each gives rise to a protein with significantly altered enzymatic
properties. In addition, the planar Trp
has been replaced
by Ile to discover the former's influence on the active site.
Since sequential comparisons have indicated dihydropteridine reductase
is a member of the class of proteins known as short chain
dehydrogenases (11) , it can also be deduced that Ala
could have some influence on the structural properties of the
reductase as this amino acid is usually a serine in the dehydrogenases.
Therefore, the Ala
Ser mutant has also been
created. Each of the mutations influences the structural and kinetic
properties of dihydropteridine reductase and the latter are illustrated
in Table 1.
Figure 1:
The structure of the reduced
dinucleotide NADH showing the pertinent interactions with
Asp, Lys
, and Asn
and the
measured bond lengths derived from the crystal structure of the binary
complex with the E. coli expressed rat dihydropteridine
reductase.
The crystal data and refinement statistics for all
mutants are given in Table 2. All mutant crystals were similar in
form to the native: orthorhombic C222 with a = 50.10 Å, b = 139.13 Å, and c = 64.93 Å. Structures were refined to an R
18%. There was very little overall structural change between
the native and mutant proteins; however, significant changes occurred
around the particular amino acid that was mutated. In particular, a
difference of bound water molecules was noted in the active site.
It was necessary to devise a series of omit maps for the various mutants in order to confirm that the specific insertions had occurred and to measure the changes in electron density associated with the replacement amino acids.
Figure 2:
a
and f are difference maps computed with the mutant diffraction
data and the wild-type coordinates. b-e are omit maps
computed excluding the appropriate side chains from the wild-type
model. a, Y146F difference map. b, Y146F omit map. c, Y146F/K150Q omit map at residue 150. d, Y146H omit
map. e, W86I omit map. f, A133S difference map.
Contours are drawn at 3 (c, e, and f),
-3
(a) and 2.5
(b and d)
levels.
Figure 4:
Stereoview of the Tyr
Phe/Lys
Gln mutant active site showing bound
pteridine and NADH and the hydrogen bonding network around glutamine
150. Water molecule 320 is clearly shown hydrogen bonded to both
pteridine and ribose 2` hydroxyl groups, with Gln
in the
mutant somewhat more distant from the NADH than Lys
in
the wild-type structure. Water molecule 320 and selected amino acids
are numbered. Potential sites for hydrogen bonding are illustrated by broken lines.
As was
described earlier(27) , Asp resides at the end of
a typical Rossman fold that exhibits close affinity for the adenine
ribose component of NADH. The crystal structure of the native enzyme
shows strong hydrogen bonds between the distal aspartate carboxyl and
the 2`- and 3`-hydroxyl groups of the ribose unit (Fig. 1).
Consistent with the loss of these hydrogen bonds in the Asp
Ile mutant is the lower affinity exhibited by this mutant
compared to wild type for NADH (K
= 0.35
µM compared with K
= 0.024
µM). Clearly this is an order of magnitude transition with
an associated significant fall off in k
to 36
s
, demonstrating only 23% of the wild-type activity.
Progressing along the NADH molecule, from right to left in Fig. 1, Lys
interacts with the
nicotinamide ribose. Mutations to Gln, Ile, and Met demonstrate the
importance of this linkage and K
values from 0.17
to 0.35 µm were measured and k
values from
59 to 17% of wild type. A final important interaction occurs between
Asn
and the carboxamide of the nicotinamide. The
conversion of this amino acid to alanine in dihydropteridine reductase
explores this feature. So far this mutant has proven too unstable to
purify.
Because of difficulties associated with the practical
isolation and crystallization of a dihydropteridine reductase ternary
complex, the issue has been resolved by the graphical introduction of
quinonoid dihydrobiopterin into the active site(25) . This
model is based on the premise that the N5 position of the pteridine is
the recipient of the hydride transfer from NADH (29, 30) and the probability that C4 of the
nicotinamide and N5 of the pteridine are within 3.3 Å of each
other (5) . When these restrictions are applied it is apparent
from crystal structure data that Tyr is within hydrogen
bonding distance of the C4 oxygen of the pteridine. Previously it has
been hypothesized that tyrosine might be the compensatory proton donor
for the hydride transfer from the reduced dinucleotide(10) ,
therefore mutation of this amino acid to phenylalanine should have
serious consequences for the enzyme. In fact k
drops to 1.1% of the wild-type value when this change is
introduced. Interestingly, the K
for NADH is only
marginally altered and K
values for both
substrates, although lower, probably contain a greater error factor
that reflects the difficulty of measuring the low specific activity
exhibited by the protein by the usual assay. The second of these
mutations Tyr
His is somewhat more active as might
be expected a priori as histidine can itself be a proton
donor. k
rises to 15% of wild type, K
values for both substrates are normal, and the K
for NADH is virtually that of wild type.
Mutation of Trp
to Ile removes the large planar steric
interaction that sandwiches the substrate in the active site and the
kinetic results alter accordingly. The K
for NADH
is unaltered, but the k
falls to 42% of
wild-type and the K
for the pteridine substrate
rises to twice the usual figure.
Surprisingly, the double mutant
Tyr
Phe/Lys
Gln gives a
considerable rise in k
to 50% of the wild-type rate when
compared with the Tyr
Phe mutant alone. The K
for NADH in this mutant is comparable with that
of the Lys
Gln single mutant and K
values for both substrates are little altered. The Ala
Ser mutation gave no unusual results for this enzyme. The k
was 97% of wild type, the K
for NADH was marginally altered and the K
values for both substrates were similar to the wild type.
The
pH activity profiles for each of the mutants except, Tyr
His, were similar having a single peak of activity at
approximately 7 with lower rates toward higher and lower pH values (Fig. 3). Using the standard assay, results lose their accuracy
below pH 6 because of the high rate of acid catalyzed NADH
decomposition and at greater than 10 because of an erratic blank rate
probably caused by quinonoid dihydropteridine decomposition. Only the
Tyr
His mutant shows an altered profile. A clear
shoulder appears at pH 8.5 in addition to the peak of activity at pH 7.
Figure 3:
The
pH profiles of enzymatic activity. All mutants, except Tyr
His, have the general profile illustrated for the
wild-type enzyme (A) (
). Tyr
His is
shown on the same scale (
) and enlarged in B (
).
Of the many mutants examined kinetically, five have been
crystallized and structurally characterized: Trp
Ile, Tyr
His, Tyr
Phe,
Tyr
Phe/Lys
Gln, and
Ala
Ser. Asp
Ile, the various
single mutants at 150, and Asn
Ala proved
extremely unstable to purification. When in the presence of a
5-10-fold molar excess of NADH they remained stable at low
temperature in solution. Unfortunately, low yields ensued from
chromatography unless buffer solutions contained very high
concentrations of the dinucleotide, making the practical necessity of
repeated purifications prohibitively expensive. Nevertheless,
sufficient quantities of the Lys
Gln, Lys
Ile, and Lys
Met, but not the
Asn
Ala mutants were isolated for rapid
measurements of kinetic constants. Activity was lost, however, too
rapidly for the isolation of sufficient purified material to allow
x-ray crystallography. This observation suggests the hydrogen bonds
connecting Asp
, Lys
, and Asn
to the two ribose components and carboxamide of the dinucleotide (Fig. 1) are of prime importance for securing the correct
orientation and binding of NADH to the protein backbone.
The loss of
activity exhibited by the single mutant Tyr
Phe
has been attributed to the loss of the phenolic tyrosine OH. Structural
alignment of Tyr
and Lys
suggest
interactive forces might exist of sufficient magnitude to allow
liberation of the phenolic hydrogen as a proton and thus provide the
compensatory proton transfer required to match the NADH hydride
donation occurring to the pteridine in the reductive
process(10) . Analogous transfers have been suggested in the
case of aldose reductase (31) and the
dehydrogenases(32) .
Surprisingly, the double mutant
Tyr
Phe/Lys
Gln was also
sufficiently stable to allow purification and crystallization (Fig. 4). This is difficult to understand as the Lys
Gln change might suggest similar instability to the single
mutant would occur. Probably, the introduction of the second mutant,
Tyr
Phe, must alter the orientation of the various
functional groups in this region of the molecule. This conclusion for
the enhanced double mutant binary complex stability is supported by the
observation that loss of the phenolic OH of the tyrosine in the single
Tyr
Phe mutant causes a profound 2 orders of
magnitude change in k
(Table 1), whereas,
in contrast, the double mutant regains considerable activity to give up
to 50% of the wild-type response. Examination of the active site of the
double mutant (Fig. 4) suggests that the altered position taken
by H
O 320 might be a major contributor to both the enhanced
stability and increased activity of the double mutants. Upon substrate
binding, this water molecule is in a position to hydrogen-bond with the
carbonyl O4 of the pterin and thus is capable of transferring a proton
to the substrate. It is of interest that this water molecule 320 is
present in a similar position in all of the mutants except Tyr
His. In each case, the water is bound to the nicotinamide
ribose hydroxyl. The B-factor for the H
O 320 site is
30 Å
, except in the case of the double mutant,
in which it has a B-factor of
10 Å
. In
Tyr
Phe, only half occupancy gives H
O
320 a B-factor of
10 Å
. This suggests that the
substitution of a lysine by a glutamine in the double mutant causes a
local environmental change in which water is bound more strongly. This
apparent increase in stability of this water molecule appears
significant as it is correlated with the recovery of activity by the
Tyr
Phe/Lys
Gln double
mutant.
When Tyr is replaced by a histidine, only a
2-fold higher change in K
is observed for NADH,
relative to wild type; however, there is a considerable loss of
activity (i.e.
85%). This probably reflects the change of
proton donor from tyrosine to histidine. Although, histidine should be
a better proton donor, the hydrogen bond geometry with tyrosine and
histidine at the active site is different. As seen in Fig. 5,
the substrate can bind in a position such that the O4 of the substrate
forms a hydrogen bond with the histidine, but the angle of proton
transfer is less than ideal. Moreover, the histidine interaction could
be more complex. Just as the pK of the tyrosine proton is
affected by the proximity of the protonated amino group of lysine 150,
so could the protonation of the histidine imidazole be affected. In
addition, it is apparent in Fig. 5that the 2-amino substituent
and 3-nitrogen of the quinonoid dihydrobiopterin substrate are near
water molecule 263. The delocalization of the quinonoid pteridine
electronic structure is such that the compensatory protonation to
hydride transfer could occur at the 2-, 3-, or
4-positions(33) . Therefore, in the case of the histidine
mutant, this altered reaction pathway might occur to afford the 15%
residual activity and may also contribute to the altered pH profile
shown in Fig. 3. The water molecule 263 is present in all the
other structures, except the Ala
Ser mutant, but
it would appear that in these instances protonation is favored to occur
from the tyrosine hydroxyl or its aqueous replacement. It is not
inconceivable a minimal contribution from this alternate proton source
might occur with other mutants. It is interesting to note that when the
tyrosine proton donor occurring at the active site of aldose reductase
is replaced by histidine, compensatory movements of molecular water are
observed (31) .
Figure 5:
A, scheme illustrating the unfavorable
hydrogen bonding interaction between pteridine substrate and histidine
146 in the Y146H mutant. R is the dihydroxypropyl substituent
of biopterin. Top, wild-type model substrate interactions with
the His of the Tyr
His mutant superimposed. Bottom, model substrate interaction comparing Phe and His at
146. With Phe at 146, H
O 320 is present, but with the His
mutant this water molecule is not present at this position. B,
stereoview of the hydrogen bonding network associated with water
molecule 263 that could offer alternate proton sources for completion
of the reductive process. Nomenclature is as described in the legend to Fig. 4with additional selected nitrogen and oxygen sites on the
protein backbone being emphasized for clarity and hydrogen bond lengths
marked in Å.
It is apparent from the orientation of
Trp relative to the active site (5) that
considerable interaction is possible between this planar substituent
and the planar pteridine substrate. The tryptophan side chain keeps the
substrate pinned against the nicotinamide ring. It is therefore not
surprising that the generation of the Trp
Ile
mutant leads to a protein with 42% comparative reactivity. The crystal
structure (Fig. 6) demonstrates graphically the differential
interaction of the isoleucine and tryptophan side chains with the
pteridine molecule. It is interesting to note also that, although not a
direct measure of affinity, the K
value of the
pteridine substrate rises to 70 µM, a figure that is 2.5
times the wild-type figure, reinforcing the concept that this mutant
enzyme is less efficient in its pteridine reductive capacity. Clearly
Trp
plays a significant role in creating the correct
contours for the ternary complex active site.
Figure 6:
Stereoview of the Trp
Ile mutant structure showing the altered interaction of this amino acid
side chain with the pteridine substrate. For comparison, the wild-type
structure has been superimposed at this position. The nomenclature is
as described in the legend to Fig. 5.
The Ala
Ser mutant is an interesting variant. The serine
substitution is present in a majority of the known short chain
dehydrogenases (7, 11) and the
UDP-epimerase(34) . With dihydropteridine reductase, when this
substitution is made, little alteration in kinetic properties relative
to wild type are observed. Although Ser
hydrogen bonds to
two backbone nitrogens (Fig. 7), it is apparent that the
hydroxyl group of Ser
can impinge on the active site. The
position of Ser
in Ala
Ser is
consistent with the position of Ser
in
3
,20
-hydroxysteroid dehydrogenase(28) . In the case
of the dihydropteridine reductase reaction the availability of yet
another proton source is unnecessary; however, in the case of the
dehydrogenases where a proton sink is required, this serine molecule
and its hydrogen bonding network may play an important role in
facilitating proton transfer and thus aid the overall oxidative
dehydrogenation that occurs in many of these reactions.
Figure 7:
Stereoview of the Ala
Ser mutant structure showing the hydrogen bonding network created when
a serine is introduced at this position. Also shown are additional
interactions created to the backbone of amino acids 134 and 135. The
nomenclature is as described in the legend to Fig. 5b.
It is
apparent from these results that the dihydropteridine reductase dimer
is very sensitive to the correctly oriented binding of the reduced
dinucleotide cofactor. In particular the associations of
Asp, Lys
, and Asn
are
prerequisites for a functional enzyme. Moreover, without dinucleotide
present the protein rapidly loses its active conformation as is
observed by the irreversible loss of activity during purification and
dialysis. It is probable that a protonated Lys
is also
important for the rapid elimination of NAD
after
reduction has occurred. Such a function can be inferred from the NMR
experiments carried out with the analogous epimerases(8) ,
where a polarization of the nicotinamide positive charge throughout
this region of the oxidized dinucleotide is observed in suitably
N-labeled NAD
derivatives. Based on the
extensive hydrogen bonds around Lys
, it will undoubtedly
be charged in dihydropteridine reductase and most probably in the
steroid dehydrogenases where similar configurations have been
reported(28, 35) . The
-amino group of
Lys
in dihydropteridine reductase protonated at the pH
optimum of the reaction will thus diminish a potential charge-charge
interaction by the ejection of NAD
after reduction of
substrate has occurred. The K
for NAD
by the wild-type enzyme is
200 µM,
demonstrating the enzyme's low affinity for the oxidized form of
the dinucleotide. Because of the instability of the Lys
and Asn
mutants, particularly in the absence of
cofactor, it has not yet been possible to measure the K
values of NAD
with these mutant enzymes. The
point of attachment via Asn
, where hydrogen bonding
secures the carboxamide substituent of the nicotinamide and ensures the
correct orientation of the pro-S hydrogen of NADH toward the incoming
pteridine, gives rise to the classification of dihydropteridine
reductase as a B-stereospecific dehydrogenase (36) . With a
mutation at 186 from asparagine to alanine, the nicotinamide portion of
the NADH cannot be rigidly orientated for proper reduction of the
substrate.
The mechanistically important function of Lys to activate Tyr
is clearly lost in all the mutants
occurring at both the 146 and 150 positions as is evidenced by the
markedly lower k
values exhibited by these
mutants. The most extreme changes occur with Tyr
Phe and Tyr
His. The compensatory positioning of
water molecules in the vicinity of the substrate and the possibility
that protonation could occur at the 2-, 3-, or 4-position of the
quinonoid pteridine, described above, could explain, however, the
residual activity observed with these mutants.
Clearly, until NADH
is bound, no active site exists with this enzyme, which supports the
ordered kinetics reported by previous workers(37) . Of
considerable importance toward completing the creation of this site is
Trp. It allows a planar aperture to form in the molecule
that accommodates the sandwiching of the quinonoid dihydropteridine
substrate at the site thus holding the molecule during the reductive
process. After reaction the enzyme then rapidly ejects both product and
NAD
and prepares for a further cycle. The instability
of the protein minus dinucleotide suggests that if intracellular
concentrations of the reduced dinucleotide are low the enzyme will have
a short half-life. This will be particularly so if mutations are
present at positions 37, 150, or 186.