From the Graduate School of Biological
Sciences, Nara Institute of Science and Technology, Ikoma, Nara
630-0101, Japan, the ¶ Institute for Chemical Research, Kyoto
University, Uji, Kyoto 611-0011, Japan, and the
Faculty of
Biotechnology, Fukui Prefectural University, Matsuoka-cho, Yoshida-gun,
Fukui 910-1195, Japan
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ABSTRACT |
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Two tropinone reductases (TRs) constitute a key
branch point in the biosynthetic pathway of tropane alkaloids, which
are mainly produced in several solanaceous plants. The two TRs share
64% identical amino acid residues and reduce the 3-carbonyl group of a
common substrate, tropinone, but they produce distinct alcohol products
with different stereospecific configurations. Previous x-ray
crystallographic analysis has revealed their highly conserved overall
folding, and the modeling of tropinone within the putative substrate-binding sites has suggested that the different
stereospecificities may be determined solely by the different binding
orientations of tropinone to the enzymes. In this study, we have
constructed various mutant TRs, in which putative substrate-binding
residues from one TR were substituted with those found in the
corresponding positions of the other TR. Substitution of five amino
acid residues resulted in an almost complete reversal of
stereospecificity, indicating that the different stereospecificities
are indeed determined by the binding orientation of tropinone. Detailed
kinetic analysis of the mutant enzymes has shown that TR
stereospecificity is determined by varying the contributions from
electrostatic and hydrophobic interactions and that the present TR
structures represent highly evolved forms, in which strict
stereospecificities and rapid turnover are accomplished together.
Two tropinone reductases
(TRs)1 catalyze key branch
point steps in the biosynthesis of tropane alkaloids, which include
several medicinally important compounds such as hyoscyamine (its
racemic form, atropine) and scopolamine (1). The two enzymes share a
common substrate, tropinone, and transfer the pro-S hydrogen atom of the cofactor NADPH to the 3-carbonyl carbon atom of the substrate. However, the resulting alcohol products have opposite configurations at the hydroxyl group, i.e. one enzyme, TR-I
(EC 1.1.1.206), produces tropine with a 3
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxyl group, whereas the other, TR-II (EC 1.1.1.236), produces pseudotropine (
-tropine) with a
-hydroxyl group (Fig. 1).
Tropine and
-tropine are not interconverted in vivo (2)
and are further metabolized to various alkaloid products.
View larger version (22K):
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Fig. 1.
Reactions of TRs. Carbon atoms of the
tropane ring system are numbered in tropinone.
cDNAs encoding TRs have so far been isolated from two plant
species, Datura stramonium and Hyoscyamus niger,
both of which belong to the family Solanaceae (3,
4).2 All four TRs have been
expressed in Escherichia coli as native proteins, and gel
filtration experiments with these recombinant TRs have revealed that
the enzymes of D. stramonium exist as a homodimer, whereas
the TRs of H. niger most likely exist as a homotetramer
(5).2 In both species, TR-I and TR-II share 64% identical
amino acids from the 260-274 residues comprising each subunit,
suggesting that the two enzymes diverged relatively recently from the
same ancestral protein. The amino acid sequences also indicated that the TRs belong to the short chain dehydrogenase/reductase family (6).
The most intriguing questions about the TRs ask how these enzymes have
evolved to catalyze opposite stereospecific reductions of the same
substrate, and what molecular mechanism enables these homologous
enzymes to control the stereospecificity in such a strict manner? A
clue to answering these questions was first obtained from the analysis
of a series of chimeric TR enzymes, which suggested that the different
stereospecificity was conferred by a structural difference in their
substrate-binding sites (5). Recent x-ray crystallographic analysis of
the TRs from D. stramonium has revealed that the two enzymes
share almost identical overall protein folding and that both the
cofactor-binding site and the catalytically important Tyr residue are
structurally very well conserved within their three-dimensional
structures (7). Well conserved positions for both the hydride donor
(NADPH) and the proton donor (catalytic Tyr residue) have suggested
that the configurations of the reaction products (3- or
3
-hydroxyl group) are determined solely by the binding orientations
of tropinone to the enzymes (7). Furthermore, the predicted
substrate-binding site of each enzyme appears to be consistent with the
predicted binding orientation of tropinone that produces the hydroxyl
group with the relevant configuration (Fig.
2, A and B) (7).
The substrate-binding sites of the TRs have different electrostatic
charge distributions that may fix opposite orientations for tropinone;
in TR-I, the positive charge of His112 may repulse the
positive charge of the tropinone nitrogen atom, whereas in TR-II, the
favorable interaction between the negative charge of Glu156
and the positive charge of the tropinone nitrogen atom may fix the
opposite binding orientation. In addition, the putative
tropinone-binding sites of both TRs were composed mostly of hydrophobic
amino acids, some of which were replaced with different amino acids in
TR-I and TR-II. Such changes may differentially shape the
substrate-binding sites and thereby aid in fixing the binding
orientation of tropinone. Although bound tropinone is predicted to be
surrounded by 11 amino acid residues, comparison of the four TR
sequences (two TRs from the two plant species) has revealed that only
five residues can be correlated with stereospecificity (Fig.
2C) (7).
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A potential method to confirm these hypothetical tropinone binding
modes would be the crystallographic analysis of the
protein-cofactor-tropinone ternary complexes. However, such complex
crystals cannot be obtained in all cases, and even if successful, the
structure alone is not sufficient to understand the role of each amino
acid residue. Site-directed mutagenesis is another method that could be
used to confirm the hypothetical tropinone binding mode and thereby compensate for the present absence of crystallographic studies of the
ternary complexes. Here we report the construction and characterization
of a series of mutant TR enzymes. Substitution of the five amino acids
involved in the putative TR substrate-binding sites was found to be
sufficient to switch their stereospecificities. The results confirmed
most of the predicted mechanisms controlling the different TR
stereospecificities and further revealed different contributions from
electrostatic and hydrophobic interactions to substrate binding.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis-- Mutations were introduced into the TR-coding regions of the expression vectors, pETTR1 and pETTR2, which respectively express the native TR-I and TR-II enzymes of D. stramonium under the control of the T7 promoter (7). Mutagenesis was performed by the "splicing by overlap extension" method (8), using two complementary primers that contain the desired mutation(s) and two other primers that anneal to either upstream or downstream regions of the vector (T7 promoter or T7 terminator primers). Amplified DNA from the second round of polymerase chain reactions was digested at the XbaI and EcoRI sites derived from the multicloning site of the pET21d vector. The digested DNA was purified by a Chroma Spin 400 column (CLONTECH) and ligated into new pET21d vector at the XbaI and EcoRI sites, so as to reconstruct the vector sequences. Because the mutagenesis was performed using many cycles of polymerase chain reactions with Taq polymerase, all the mutated plasmids were sequenced, and only plasmids without unintended missense mutations were used to express proteins.
Protein Expression and Purification--
Mutant and wild-type TR
enzymes were expressed in E. coli strain BL21 (DE3) and
extracted as described previously (7). Buffer-soluble proteins were
fractionated by precipitation with ammonium sulfate. Proteins
precipitating at 45-75% saturation of ammonium sulfate were dissolved
in buffer A (10 mM potassium phosphate, pH 7.0, 1 mM dithiothreitol, 10% (v/v) glycerol) containing 35%
saturation ammonium sulfate and loaded onto a butyl-Sepharose fast flow
column (1.5 × 0.9 cm, Amersham Pharmacia Biotech). Proteins were
eluted with a stepwise reduction in the ammonium sulfate concentration
of the buffer. Fractions enriched with TR proteins were pooled and then
purified further by affinity chromatography. For both the wild-type and
mutant TR-I, the butyl-Sepharose fraction buffer was first exchanged
for buffer B (identical to buffer A, except pH 6.4) using a PD-10
column (Amersham Pharmacia Biotech) and then loaded onto a
Red-Toyopearl 650 ML column (0.9 × 2.0 cm, Toso, Japan). Bound TR
protein was eluted with a stepwise gradient of NaCl (0-1.5
M) in buffer A. For the wild-type and mutant TR-II, the
butyl-Sepharose fraction buffer was exchanged for buffer A, loaded onto
a 2',5'-ADP-Sepharose column (0.9 × 2.7 cm, Amersham Pharmacia
Biotech) and eluted with a stepwise gradient of KCl (0-1.0
M) in buffer A. Affinity chromatography fractions were
desalted by PD-10, aliquoted, and stored at 80 °C until further use.
Enzyme Assay and Kinetic Analysis--
TR activity was measured
by either product quantification with gas-liquid chromatography or
photometric measurement of NADPH consumption (9). For kinetic analysis,
the photometric method was used because of its accuracy in measuring
the steady state velocity of the reaction. Km and
kcat values were determined from [S]
versus [S]/v plots using at least five
different [S] values, where [S] is the concentration of the
substrate and v is the activity at each substrate concentration.
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RESULTS |
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Construction of Mutant TR Enzymes--
A previous x-ray
crystallographic study predicted 11 amino acid residues from each TR
that would possibly come in contact with tropinone bound to the enzyme.
Comparison of the amino acid sequences of TR-I and TR-II from the two
plant species revealed that only five of these 11 residues could be
correlated with stereospecificity, i.e. the five positions
are substituted by different amino acids according to enzyme type but
are conserved across the species (Fig. 2C) (7). These five
residues were classified into two groups: one having different
polarities between TR-I and TR-II and the other conserving
hydrophobicity. The former group (group A) has three residue pairs:
His112-Tyr100,
Ala160-Ser148, and
Val168-Glu156 (where the order is TR-I-TR-II),
whereas the latter (group B) has two residue pairs:
Ile223-Leu210 and
Phe226-Leu213. These five positions are
hereafter, respectively, referred to as A1, A2, A3, B1, and B2 (Fig.
2C). In this study, mutations were first made to
individually exchange the three group A residues between TR-I and
TR-II, whereas the two group B residues were exchanged together. These
mutations were then combined in a single enzyme in various combinations
to produce more mutated TR enzymes. The mutant enzymes were expressed
in E. coli using the T7 expression system (10). As for the
wild-type TRs, the mutant enzymes were expressed at very high levels,
with the TR-I mutants showing greater expression than the TR-II mutants
(Fig. 3A). Each of the
expressed TR proteins was purified by ammonium sulfate precipitation,
followed by hydrophobic interaction chromatography. The final stage of purification involved chromatography using either a Red-Toyopearl or a
2',5'-ADP-Sepharose column for TR-I and TR-II, respectively. Mutant TR
enzymes were purified under fundamentally identical conditions to those
used for the corresponding wild-type enzymes (data not shown). These
two chromatographic steps resulted in nearly homogenous TR protein
preparations (Fig. 3B).
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Purified enzymes were first analyzed for their affinity toward tropinone and NADPH (Table I). Because the affinity between TR-I and tropinone has been reported to be dependent on reaction pH (11), the following analyses were performed under a physiologically relevant condition, namely pH 7.0 at 28 °C, unless noted otherwise. Whereas most of the mutant enzymes showed several orders of magnitude higher Km values for tropinone as compared with the corresponding wild-type enzymes, affinity for NADPH was not very much affected by any mutation(s), with their Km values remaining within the same order of magnitude as those of the wild types. This observation reflects the fact that mutations were made only to putative tropinone-binding sites. Their relatively constant affinity for NADPH, together with their similar behavior during purification, indicates that the mutant enzymes retained the wild-type tertiary structure.
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Stereospecificities of Mutant Enzymes--
Stereospecificity of
the mutant enzymes was analyzed by quantifying the reaction products,
tropine and -tropine, by gas-liquid chromatography. Because the
mutant enzymes had differing affinities for tropinone, as seen in Table
I, enzyme reactions were performed at four different tropinone
concentrations (0.05, 0.5, and 5 mM, plus the
Km of each enzyme).
Fig. 4 shows the TR-I/TR-II activity
ratios measured as described above. Among the single mutants of the
three A residues, only the mutation at A3 affected stereospecificity.
In TR-II this position is occupied by Glu, an acidic residue predicted
to be the most important determinant of tropinone binding orientation.
When mutations at residues A1 and A2, which alone had no effect on
stereospecificity, were added to the above A3 mutants,
stereospecificity was shifted in a cumulative fashion. At a tropinone
concentration of 5 mM, the stereospecificity of the triple
mutants of both TR-I and TR-II was shifted by more than 90%. At lower
tropinone concentrations, however, the stereospecificity shift was
diminished, especially for the TR-II mutant, which possessed two
productive tropinone binding orientations, with the one producing
-tropine (TR-II orientation) still being of higher affinity.
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In both TR-I and TR-II, substitution of the B residues alone had no effect on stereospecificity. When these group B mutations and the group A triple mutations were combined in a single enzyme to give quintuple mutants, the stereospecificity shifts were further enhanced, and the dependence on tropinone concentration seen in the triple mutant disappeared.
Role of His112 in TR-I Catalysis--
The
results obtained above disagreed with our previous prediction
concerning the role of His112 in TR-I. The positive charge
that would arise from His112 seems to have no direct
contribution to stereospecificity, because the H112Y mutant retained
wild-type TR-I stereospecificity. To understand the role of
His112 in TR-I catalysis, we constructed an additional
mutant, H112F, in which His112 was replaced with Phe, an
amino acid with similar size but higher hydrophobicity. This mutant
retained TR-I stereospecificity, as expected from the H112Y mutant
(data not shown), and its affinity for tropinone was also similar to
that of the H112Y mutant (Table I). However, the H112F mutant showed a
reduced turnover rate, with kcat values at pH
7.0 being 20 and 31% of the wild-type and the H112Y mutant,
respectively (Table I). These observations suggest that although the
positive charge of His112 was dispensable for
stereospecificity, some degree of polarity at this position is
necessary for efficient TR-I catalysis (and possibly for TR-II as
well). This was confirmed by analyzing the kinetic parameters of the
mutants at various pH values. Both H112Y and H112F mutants lost the pH
dependence of kcat values that was observed in
the wild-type TR-I, especially the sharp increase in
kcat at acidic pH (Fig.
5A). Conversely, the Y100H
mutation conferred a pH dependence to the kcat
values of TR-II (Fig. 5B). Given that the
pKa of the His side chain is about 6.4 (12), the
observed pH dependence of the kcat values likely
reflects the change in hydrophobicity of His112.
Tyr100 of TR-II (pKa 9.7) is thought to
be protonated within the pH range tested, but the -hydroxyl group
may still exhibit sufficient polarity. These results indicate that some
degree of polarity at this position is necessary for efficient
catalysis by both TRs.
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Both H112Y and H112F mutants showed an increase in
Km values at acidic pH similar to wild-type TR-I
(Fig. 5C), indicating that the low affinity between TR-I and
tropinone at acidic pH is not because of unfavorable contact between
the charged His112 residue and the hydrophobic C6-C7 bridge
of tropinone but rather because of unfavorable contact between the
charged tropinone nitrogen atom and the hydrophobic surface of TR-I
(Fig. 2A). In contrast to TR-I, the Km of
TR-II was altered to be pH-dependent by the Y100H mutation
(Fig. 5D). This is consistent with our binding model, where
the nitrogen atom of tropinone comes in close contact with
Tyr100 of TR-II (Fig. 2B). At acidic pH, a His
residue in place of Tyr100 would introduce an unfavorable
repulsion toward the nitrogen atom of tropinone bound in the wild-type orientation.
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DISCUSSION |
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Mechanisms Controlling Stereospecificity-- From the x-ray crystallographic analyses of the TR-I-NADPH complex crystal and the TR-II protein crystal, it was predicted that the different stereospecificities of TRs are determined by the binding orientations of tropinone in the enzymes (7). Comparison of TR substrate-binding sites revealed only a few amino acids that were selectively different according to enzyme type, raising the possibility that TR stereospecificity may be determined by a relatively small number of amino acids. In this study, a series of site-directed mutagenesis experiments proved those predictions to be relevant; by exchanging five amino acid residues between TR-I and TR-II, stereospecificity was reversed by more than 90% irrespective of substrate concentrations. It should be noted, however, that these residues were not solely responsible for binding tropinone with high affinity and reactivity. The quintuple mutants of both TR-I and TR-II showed relatively high Km values for tropinone and in the case of TR-I, a decreased kcat value as well. Therefore, an additional substitution(s) is necessary to attain reversed stereospecificity together with a rapid turnover rate. A candidate for such a residue is Lys214 of TR-I, which is missing from TR-II (Fig. 2C). This residue is positioned at the edge of the small lobe, a domain that constitutes one side wall of the substrate-binding cleft. Deletion of this residue from TR-I (as in TR-II) would result in the displacement of the two side walls of the cleft and may alter the relative orientations of the tropinone-binding residues. There may be more such residues that indirectly affect substrate binding.
Although TR-I and TR-II have an almost identical tertiary structure and use a subset of amino acids that occupy similar three-dimensional positions, they employ different strategies to hold tropinone in its correct orientation. In TR-II, the binding orientation is considered to be fixed primarily by the favorable electrostatic interaction between Glu156 and the nitrogen atom of tropinone, with the other amino acids helping to increase binding affinity and fine-tuning binding orientation. Without these favorable electrostatic interactions, the hydrophobic amino acids of the TR-I substrate-binding site provide a more complementary fit to tropinone than does TR-II. Consistent with this prediction are the results obtained from the group B mutations. The I223L/F226L mutations impaired the affinity of TR-I for tropinone, with the Km value of the mutant being 5.11 mM (a 44-fold increase from the wild-type), whereas the Km of the L210I/L213F TR-II mutant remained low at 0.0448 mM (Table I), indicating these two hydrophobic residues to be relatively more important for substrate binding in TR-I. Furthermore, among the 11 residue pairs that were predicted to form the TR substrate-binding sites, in TR-I, only one pair shows any interspecies variety (Val208-Ile), whereas there are three such residue pairs in TR-II (Val147-Ile, Val153-Leu, and Leu196-Met) (Fig. 2C). The diminished variety of TR-I residues may reflect the importance of these noncharged amino acids for the high affinity binding of tropinone to TR-I.
The different substrate recognition mechanisms of TR-I and TR-II raise the question as to why TR-I does not use electrostatic interaction to increase its affinity for tropinone. To address this question, we introduced either Glu or Asp in place of Val203 in TR-I, this being the only possible position where mutagenesis to an acidic residue would provide an electrostatic interaction with the tropinone nitrogen atom. These mutants showed both lower affinity for tropinone and a reduced turnover rate (data not shown). Moreover, a computer modeling experiment demonstrated that neither Glu nor Asp (in place of Val203) was able to locate its side chain carboxyl group at a distance favorable to the tropinone nitrogen. These observations suggest that the TR backbone does not permit TR-I to use an electrostatic interaction in binding tropinone in a correct and productive orientation.
Implication for the Evolution of TRs--
The impaired
stereospecificity and reactivity (as measured by
kcat/Km values) observed in
all the mutant enzymes indicate that the wild-type structures of both
TRs represent highly evolved forms. For instance, His112 of
TR-I, which appeared to have no direct contribution to
stereospecificity, was, however, found to be important for both high
affinity binding of tropinone and rapid turnover. The corresponding
TR-II residue, Tyr100, cannot replace His, because this
mutation reduces the affinity for tropinone, and no increase in
kcat can be expected at physiological pH (Fig.
5B). This could mean that the metabolism of tropinone may
have evolved under strong selective pressure, i.e. the
regulated production of two different stereoisomers might have
conferred selective advantage to the plants producing them. In H. niger, tropine is esterified to an organic acid to give
hyoscyamine and then scopolamine (Fig. 1) (1). -Tropine can also be
esterified to different organic acids, but most
-tropine is
considered to be polyhydroxylated to give calystegines (Fig. 1) (13).
Whereas hyoscyamine and scopolamine act on the nervous system of
mammals, calystegines have a glycosidase-inhibiting activity and can
also serve as nutritional mediators in the rhizosphere (14, 15). Different functions of the tropine- and
-tropine-derived alkaloids might have prompted the acquisition of separate TR enzymes to control
the metabolic flow at this branching point.
Recent random cDNA sequencing projects have revealed that genes
highly homologous to TRs are widely distributed in the plant kingdom.
We have determined the full-length sequences of five such clones from
Arabidopsis and found that they encode proteins with high
homology to TRs along the entire polypeptide chain (47-54% identical
residues) but have a different set of predicted substrate-binding residues.2 This indicates that the same protein backbone is
also utilized for enzymes that function in different metabolic
pathways. The original TR enzyme must have arisen from a common
ancestor with those enzymes via mutations to the substrate-binding
site. This original TR enzyme likely had random stereospecificity,
because our mutation experiments indicated that a strict
stereospecificity can only be achieved after fine-tuning the
substrate-binding structure, which requires mutations at several amino
acid residues. After gene duplication, each of the progeny TRs may have
evolved strict and opposite stereospecificities. An alternative
evolutionary process can also be assumed. Because the stereospecificity
of most of the TR mutants was dependent on tropinone concentration, the
ancestral TR that potentially had a random stereospecificity might have
produced only one stereoisomer, provided that only low concentrations
of tropinone were present during the early stages of evolution.
Therefore, it is possible that an ancient TR first evolved to have
strict stereospecificity for one configuration, and the opposite
stereospecificity was later acquired by switching its stereospecificity
using a process similar to this mutagenesis experiment. Consistent with
this latter evolutionary process is the recent discovery of
calystegines (3-nortropane alkaloids) in well known solanaceous
plants such as potato, tomato, and sweet pepper, in which no
3
-tropane alkaloid has been reported (16, 17). Likewise, no plant
species has so far been shown to possess only TR-I activity. These
observations imply a wider occurrence of TR-II in this plant family,
and hence the later divergence of TR-I from TR-II. Exploring the TR
gene(s) in such plant species may provide insights into the emergence
of the two configurations in the evolution of tropane alkaloid biosynthesis.
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ACKNOWLEDGEMENT |
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We thank Dr. Robert Winz for critically reading the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-743-72-5482; Fax: 81-743-72-5489; E-mail: k-nakaji{at}bs.aist-nara.ac.jp.
2 K. Nakajima and T. Hashimoto, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
TR, tropinone
reductase;
-tropine, pseudotropine.
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REFERENCES |
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