From the Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany
Received for publication, July 16, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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Deoxyhypusine synthase participates in the
post-translational activation of the eukaryotic initiation factor 5A
(eIF5A). The enzyme transfers the aminobutyl moiety of spermidine to a
specific lysine residue in the eIF5A precursor protein,
i.e. eIF5A(lys). Homospermidine synthase catalyzes an
analogous reaction but uses putrescine instead of eIF5A(lys) as
substrate yielding the rare polyamine homospermidine as product.
Homospermidine is an essential precursor in the biosynthesis of
pyrrolizidine alkaloids, an important class of plant defense compounds
against herbivores. Sequence comparisons of the two enzymes indicate an
evolutionary origin of homospermidine synthase from ubiquitous
deoxyhypusine synthase. The two recombinant enzymes from Senecio
vernalis were purified, and their properties were compared.
Protein-protein binding and kinetic substrate competition studies
confirmed that homospermidine synthase, in comparison to
deoxyhypusine synthase, lost the ability to bind the eIF5A(lys) to its
surface. The two enzymes show the same unique substrate specificities,
catalyze the aminobutylation of putrescine with the same specific
activities, and exhibit almost identical Michaelis kinetics. In
conclusion, homospermidine synthase behaves like a deoxyhypusine
synthase that lost its major function (aminobutylation of eIF5A
precursor protein) but retained unaltered its side activity
(aminobutylation of putrescine). It is suggested as having evolved from
deoxyhypusine synthase by gene duplication and being recruited for a
new function.
Deoxyhypusine synthase (EC 2.5.1.46) catalyzes the first step of
the post-translational activation of the eukaryotic initiation factor
5A (eIF5A)1 (1-3). In this
two-step process (Fig. 1A),
which is one of the most specific post-translational modifications
known to date (4, 5), a specific lysine residue in the eIF5A precursor
protein is modified to a hypusine residue. Activated eIF5A is the only cellular protein known to contain this unusual amino acid; it appears
to be ubiquitously present in the Eukarya and Archaea. Hypusine
formation is coupled to cell proliferation and is essential for cell
survival, but the mode of action is still not well understood. Disruption of either the deoxyhypusine synthase gene or the eIF5A gene
in yeast leads to lethal phenotypes (6-8). Recent evidence indicates
various roles of the eIF5A in the cell, from translation to mRNA
decay to nuclear protein export (9). It has been suggested that eIF5A
may function as a bimodular protein capable of interacting with
proteins and RNA (10). RNA binding depends on both the presence of the
hypusine residue in the eIF5A and conserved core motifs of the target
RNA (11). It is supposed that the activated eIF5A in plants is of
similar importance. Recently eIF5A and the first plant deoxyhypusine
synthases were cloned and characterized from tobacco and Senecio
vernalis (12, 13). It was suggested that eIF5A in plants may be
involved in senescence-induced programmed cell death (14), as well as
early development of seedlings (15).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Reactions catalyzed by deoxyhypusine synthase
(EC 2.5.1.46) (A) and homospermidine synthase
(spermidine-specific) (EC 2.5.1.45) (B).
Deoxyhypusine synthase commences the post-translational activation of
eIF5A(lys) by aminobutylation of a specific lysine residue yielding
eIF5A(dhp), which in a subsequent reaction is hydroxylated to the
active eIF5A. Homospermidine synthase catalyzes the formation of
homospermidine. Homospermidine is the unique precursor of a
necine base, the common module of all pyrrolizidine alkaloids such as
senecionine N-oxide.
Plant homospermidine synthase (spermidine-specific) (EC 2.5.1.45) catalyzes the first pathway-specific reaction in the biosynthesis of pyrrolizidine alkaloids (for review, see Ref. 16) (Fig. 1B). The pyrrolizidine alkaloids represent a class of typical secondary compounds of vast chemical diversity with more than 360 known structures. They are constitutively expressed in species of restricted phylogenetic distribution within the angiosperms. More than 95% of the alkaloid-producing species belong to four plant families, i.e. Asteraceae (tribes Senecioneae and Eupatorieae), Boraginaceae (many genera), Fabaceae (mainly Crotalaria) and Orchidaceae (nine genera). The remaining alkaloid-containing species are found scattered in six other unrelated families (for review, see Ref. 17). The pyrrolizidine alkaloids are part of the plant's constitutive defense against insect herbivores. During their coevolutionary adaptation, a number of insect species from unrelated taxa not only learned to cope with the plant's defense barrier but developed mechanisms to recruit the alkaloids from their host plants and utilize them for their own defense against predators (16-18). It appears that the pyrrolizidine alkaloids are maintained under strong selection through herbivory. The sporadic occurrence of pyrrolizidine alkaloids among angiosperm taxa provokes the question whether pyrrolizidine alkaloids evolved once in a common ancestor of all pyrrolizidine alkaloid-producing species, followed by independent losses, or whether the biosynthetic pathways evolved several times independently in separate lineages (19).
Recently a first approach to this intriguing problem was attained (13). Cloning, sequencing, and functional expression of plant homospermidine synthase from S. vernalis (Asteraceae) revealed striking molecular similarities to ubiquitous eukaryotic deoxyhypusine synthase. The overall amino acid sequence identity between plant homospermidine synthase and deoxyhypusine synthase from yeast (20), Neurospora crassa (21), and human (22) accounts for 53, 56, and 61%, respectively (13). A 79% sequence identity was found between the two enzymes from S. vernalis strongly indicating an origin of plant homospermidine synthase from deoxyhypusine synthase by gene duplication. The close relation between the two enzymes was further supported by the observation that deoxyhypusine synthase, in addition to the aminobutylation of eIF5A(lys), like homospermidine synthase, catalyzes the formation of homospermidine from putrescine.
Here we compare the enzymatic properties (i.e. eIF5A binding
behavior, substrate kinetics, and substrate specificity) of the two
recombinant enzymes from S. vernalis. The results support molecular evidence that homospermidine synthase evolved from
deoxyhypusine synthase. Moreover, it is demonstrated that
homospermidine synthase did not acquire novel qualities but retained
all properties of deoxyhypusine synthase, except binding of the eIF5A
precursor protein.
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EXPERIMENTAL PROCEDURES |
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Radiochemicals and Reagents-- [1,4-14C]Putrescine (118 mCi/mmol) and [14C]spermidine (N-(3-aminopropyl)-[1,4-14C]tetramethylene-1,4-diamine) (115 mCi/mmol) were purchased from Amersham Biosciences. [14C]Homospermidine (N-([1,4-14C]4-aminobutyl)-[1,4-14C]tetramethylene-1,4-diamine) was synthesized as described previously (23) using recombinant bacterial homospermidine synthase (EC 2.5.1.44) from Rhodospeudomonas viridis overexpressed in Escherichia coli (24). Because the bacterial enzyme utilizes putrescine as both aminobutyl donor and acceptor, spermidine-free homospermidine could be prepared.
Expression and Purification of Recombinant
Proteins--
Full-length deoxyhypusine synthase and homospermidine
synthase cDNAs from S. vernalis were cloned
into pET-3a (Novagen) expression vectors and overexpressed in E. coli BL21(DE3) (Stratagene) as described (13). The two recombinant
enzymes were purified in a three-step procedure using DEAE-Fractogel
(Merck), phenyl-Sepharose CL-4B (Amersham Biosciences), and a Mono Q
HR column (Amersham Biosciences) as described previously (12).
The purified enzymes were bottled in small aliquots and preserved in
0.1 M glycine-NaOH buffer, pH 9.25, at 80 °C. All
studies were performed with these preparations, which, after thawing,
were used once only.
Full-length eIF5A precursor protein cDNA from S. vernalis was cloned into the pET-23b expression vector (Novagen) and expressed with a C-terminal His-Tag motive as described previously (13). The overexpressed protein was purified with Ni-NTA-agarose (Qiagen), according to the manufacturer's instructions.
Analysis of Enzyme-eIF5A(lys) Binding-- An eIF5A(lys) affinity column was prepared by binding the recombinant His-tagged eIF5A(lys) from S. vernalis as ligand to 100 µl of Ni-NTA-agarose (Qiagen). The resin was washed and equilibrated in 50 mM potassium phosphate buffer, pH 8.0, supplemented with 1 mM NAD+ and 5 mM 2-mercaptoethanol (affinity buffer). Crude extracts of E. coli cells expressing either deoxyhypusine synthase or homospermidine synthase (50-ml culture for each enzyme) were prepared in affinity buffer and incubated with the affinity resin for 1 h at 4 °C. After washing the resin with 6 ml of affinity buffer containing 5 mM imidazole to remove any non-bound protein, the resin-bound eIF5A(lys) protein was eluted with 250 mM imidazole in affinity buffer. The eluted fractions were analyzed by SDS-polyacrylamide gel electrophoresis on 15% gels.
Enzyme Assays-- The standard 100-µl assay for deoxyhypusine synthase activity (DHS assay) contained 0.1 M glycine-NaOH buffer, pH 9.25, 2 mM dithiothreitol, 0.1 mM EDTA, 10 µM eIF5A precursor protein, 10 µM [14C]spermidine (0.114 µCi per assay), 0.5 mM NAD+, and enzyme as indicated. Assays were incubated for 0.5-60 min at 30 °C. Reactions were stopped by adding 10 µl of 60 mM spermidine in 1 M potassium phosphate, pH 6.3, before adsorption to a 25-mm2 Whatman 3MM paper disc. The assay was further processed as described previously (12).
The standard 50-µl assay for homospermidine synthase activity contained 0.1 M glycine-NaOH buffer, pH 9.25, 2 mM dithiothreitol, 0.1 mM EDTA, 400 µM [14C]putrescine (0.05 µCi per assay), 400 µM spermidine, 0.5 mM NAD+, and enzyme as indicated. Assays were incubated for 0.5-60 min at 30 °C. Formation of homospermidine was followed quantitatively by radio-thin layer chromatography as described previously (25).
Separation of Polyamines by High Performance Liquid Chromatography (HPLC)-- The 14C-labeled products of the homospermidine synthase assays (HSS assays) with various substrates were analyzed as benzoyl derivatives prepared according to Ref. 26. An RP-18 column (Nucleosil 25 cm, 4-mm inner diameter; Macherey & Nagel) was applied. Elution and separation of the benzamides was achieved isocratically using the solvent mixture acetonitrile/1.5% phosphoric acid (40:60) (v/v); the flow rate was 1.0 ml per min. The benzamides were detected and quantified via parallel detection of the UV absorption at 230 nm and measuring the radioactivity with the LB-508 detector (Berthold). The retention times (Rt values, in min) for the polyamines are as follows: 1) diamines: diaminopropane, 5.2; putrescine, 5.4; cadaverine, 6.3; 1,6-diaminohexane, 9.4; 1,7-diaminoheptane, 11.3.; 2) triamines: spermidine, 8.2; homospermidine, 9.4; 3) tetraamines: spermine, 12.6; canavalmine, 13.0-13.7; homospermine, 14-15. The tetraamines usually eluted as rather broad peaks. For quantification cadaverine was used as internal standard.
Identification of Enzymatic Reaction Products--
Polyamines
were identified as N-methoxycarbonyl derivatives (MOC
derivatives) by GC-MS (27). The reaction products were prepared from
enzyme assays (total volume, 1.0 ml) containing the substrates (400 µM each), 500 µM NAD+, and 70 picokatal deoxyhypusine synthase or homospermidine synthase; assays were incubated under standard conditions in 0.1 M
glycine-NaOH buffer, pH 9.25, at 30 °C for 120 min. After protein
precipitation the amine fraction was pre-purified by ion exchange
chromatography via Dowex-50 W. After evaporation of the solvent
the residue was dissolved in 1.5 ml of 0.05 M HCl,
alkalized with 1 M Na2CO3, and
mixed with 20 µl of methyl chloroformate and incubated at room
temperature for 15 min. The MOC derivatives of the polyamines were
extracted into diethyl ether (three times, 1 ml each). The extracts
were combined, and the solvent was evaporated. The residue was
dissolved in 10 to 100 µl of dichloromethane prior to GC-MS analysis.
GC-MS was performed using a Carlo Erba 5160 gas chromatograph equipped
with a 30-m × 0.32-mm fused-silica column (DB-1) under the
following conditions: injector, 250 °C; split-ratio, 1:20; carrier
gas, helium 0.75 bar. The capillary column was directly coupled
to a Finnigan MAT 4515 quadrupole mass spectrometer.
Electron-impact-mass spectra were recorded at 40 eV.
Characteristics of the MOC derivatives of various diamines and
polyamines were as follows: diaminopropane (C3), molecular
ion ([M+]) 190, Kovats retention index (RI)
1485; putrescine (C4), [M+] 204, RI 1622; cadaverine (C5), [M+]
218, RI 1728; spermidine (C3C4),
[M+] 319, RI 2350; homospermidine
(C4C4), [M+] 347, RI
2570; 4-aminobutylcadaverine (C4C5),
[M+] 347, RI 2570; norspermine
(C3C3C3), [M+] 420, RI 2953; spermine
(C3C4C3), [M+] 434, RI 3058; canavalmine
(C4C3C4), [M+] 448, RI 3164; homospermine
(C4C4C4), [M+] 462, RI 3268. Canavalmine and norspermine, as reference compound, were synthesized according to Ref. 28.
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RESULTS |
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Purification and General Properties of Deoxyhypusine Synthase and
Homospermidine Synthase--
The two recombinant enzymes from S. vernalis were expressed in E. coli according to Ref. 13
and purified in a simple three-step protocol (12). The purification
procedure originally established for tobacco deoxyhypusine synthase
could be applied without modification to the S. vernalis
enzymes. Deoxyhypusine synthase and homospermidine synthase exhibit
identical elution profiles. The pooled enzymatically active fractions
obtained from the final Mono Q HR column gave single major protein
bands at ~41 kDa when analyzed by SDS-polyacrylamide gel
electrophoresis (Fig. 2). The pure enzyme
preparations were carefully preserved at 80 °C and used for all
subsequent enzymatic studies. Care was taken to treat the two enzymes
in precisely the same manner. Both enzymes display the same pH
dependence with an optimum at 9.25 in glycine-NaOH buffer (Fig.
3).
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Both deoxyhypusine synthase and homospermidine synthase catalyze the
formation of homospermidine by spermidine-dependent
aminobutylation of putrescine; this reaction was measured in an assay
called homospermidine synthase assay. Only deoxyhypusine synthase is
capable of converting eIF5A(lys) into eIF5A(dhp); this reaction was
measured in an assay called deoxyhypusine synthase assay. In
homospermidine synthase assays, deoxyhypusine synthase and
homospermidine synthase exhibit comparable specific activities of 767 and 784 picokatal mg1, respectively, which correspond to
turnover numbers kkat of 3.2 × 10
2 s
1. These values, calculated on the basis
of one active center per subunit, are in accordance with data obtained
previously (12, 13), although a 4-fold higher specific activity has
been reported for homospermidine synthase (i.e. 3206 picokatal mg
1) in comparison with deoxyhypusine synthase
(i.e. 737 picokatal mg
1) (13). In the
deoxyhypusine synthase assay a specific activity of 58 picokatal
mg
1 was determined for deoxyhypusine synthase
corresponding to a turnover number kkat of
2.2 × 10
3 s
1.
Enzyme-Substrate Interactions: Protein-Protein Binding and
Competition Kinetics--
Homospermidine synthase catalyzes the
formation of homospermidine with the same specific activity as
deoxyhypusine synthase but is inactive with eIF5A(lys). This may
suggest that homospermidine synthase retained the catalytic properties
of deoxyhypusine synthase but lost the ability to bind the eIF5A(lys)
protein to its surface. This hypothesis was verified by two means,
testing the abilities of the two enzymes to bind eIF5A(lys) protein and
substrate competition kinetics.
Affinity chromatography was chosen to examine the abilities of
deoxyhypusine synthase and homospermidine synthase to bind eIF5A(lys).
For this purpose crude extracts of E. coli cells expressing these enzymes were incubated under identical conditions with His-tagged eIF5A(lys) coupled to Ni(II) resin. If the enzymes are able to bind
eIF5A(lys) they should be retained by the immobilized eIF5A(lys) and
elute conjointly with the eIF5A(lys) protein in buffer containing high
concentrations of imidazole. Analysis of binding assays with deoxyhypusine synthase preparations by SDS-polyacrylamide gel electrophoresis revealed in addition to the eIF5A(lys) protein a second
band that migrated in the same manner as deoxyhypusine synthase (Fig.
4, lane 6). This corroborates
resent results with human deoxyhypusine synthase using a similar
approach (29). In contrast to deoxyhypusine synthase binding assays
performed with homospermidine synthase preparations always gave
negative results; the gel showed exclusively the eIF5A(lys) band (Fig. 4, lane 3).
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The binding studies confirmed the inability of homospermidine synthase
to bind the eIF5A(lys) protein to its surface. The following substrate
competition studies were performed to characterize the substrate
interactions at the active sites of the two enzymes. Because eIF5A(lys)
and putrescine should compete for the same active site at deoxyhypusine
synthase both substrates were tested as inhibitors against each other.
When putrescine was the variable substrate in the presence of various
fixed concentrations of eIF5A(lys), a noncompetitive inhibition pattern
resulted (Fig. 5), whereas fixed levels
of putrescine inhibited the aminobutylation of eIF5A(lys) in a
competitive manner (Fig. 6). The
noncompetitive inhibition of the aminobutylation of putrescine at fixed
levels of eIF5A(lys) indicates that the binding of eIF5A(lys) to the
enzyme surface is not affected by putrescine; enzyme molecules with
bound eIF5A(lys) are no longer accessible for putrescine. On the other
hand, the competitive inhibition of the aminobutylation of eIF5A(lys)
by putrescine indicates a direct competition of the two substrates for
the active center. The same experiment with homospermidine synthase,
i.e. the effect of fixed levels of eIF5A(lys) on the aminobutylation of putrescine revealed no inhibition at all (Fig. 7). This corroborates the result of the
binding studies and confirms that homospermidine synthase lacks the
ability to bind the eIF5A(lys) protein to its surface.
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Substrate Specificity of Deoxyhypusine Synthase and Homospermidine
Synthase--
In previous experiments with deoxyhypusine synthase (12)
it was shown that homospermidine can substitute for spermidine as
aminobutyl donor in both the deoxyhypusine and the homospermidine reaction. In the case of aminobutylation of putrescine, homospermidine acts as the aminobutyl donor of its own formation. The efficiency of
this reaction becomes evident in assays with 14C-labeled
putrescine as aminobutyl acceptor and non-labeled homospermidine as
aminobutyl donor. The two enzymes catalyze the transfer of the
C4 unit from homospermidine to putrescine with almost the same time course (Fig. 8A). If
homospermidine is replaced by spermidine as unlabeled aminobutyl donor,
labeled homospermidine is produced, accompanied by slow
time-dependent occurrence of labeled spermidine (Fig. 8,
B and C). The appearance of labeled spermidine
indicates the transfer of a labeled aminobutyl moiety of
homospermidine to 1,3-diaminopropane, released from the aminobutyl
donor spermidine. No products were formed in the presence of
[14C]putrescine as only substrate, indicating the
inability of the plant enzyme to utilize putrescine as aminobutyl
acceptor and donor like bacterial homospermidine synthase (EC
2.5.1.44), which uses either spermidine or putrescine as
aminobutyl donor (30, 31) (Table
I).
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The unusual substrate utilization of the two enzymes caused us to perform a detailed comparative analysis of their substrate specificities. In these studies the two enzymes were compared in their abilities to catalyze the aminobutyl transfer from 14C-labeled spermidine and homospermidine to a variety of diamines and polyamines. Controls with [14C]putrescine were included. The results are summarized in Table I. Diaminopropane and cadaverine, the next homologues of putrescine, are aminobutyl acceptors. Aminobutylation of these two putrescine homologues was demonstrated with labeled homospermidine as aminobutyl donor (Table I), whereas, because of the unavailability of labeled diaminopropane, the spermidine-dependent formation of spermidine could not be directly demonstrated. Higher putrescine homologues, i.e. diaminohexane and diaminoheptane, are ineffective as aminobutyl acceptors (data not shown). Unexpectedly, the two aminobutyl donors spermidine and homospermidine were found to act not only as aminobutyl donors but also as acceptors. The respective products of spermidine, i.e. canavalmine (C4C3C4), and of homospermidine, i.e. homospermine (C4C4C4), were detected as minor products in all assays but were formed in substantial amounts in enzyme assays containing only the respective triamines in absence of any other aminobutyl acceptor (Table I).
Almost identical results were obtained with the two enzymes. The only noticeable difference between the enzymes was a higher rate of canavalmine formation by deoxyhypusine synthase in comparison with homospermidine synthase (Table I).
The reaction products were identified as their MOC derivatives
by GC-MS according to their retention indices (RI values)
and molecular masses (see "Experimental Procedures").
4-Aminobutylcadaverine (C5C4) is already known
as a product of bacterial homospermidine synthase (31). The MOC
derivative of homospermine was identified by its unequivocal mass
spectrum (Fig. 9B). In the
case of aminobutylspermidine two isomers (i.e.
C4C3C4 and
C3C4C4) have to be distinguished. The crucial fragment is m/z 169 (Fig.
9A), which, according to high resolution mass spectrometry,
fits best to a molecular formula of
C9H15NO2 (molecular mass
(calculated), 169.110; molecular mass (measured), 169.112). This or
homologous fragments occur only in fragmentation patterns of MOC
derivatives of tetraamines with a central C3 fragment
(e.g. norspermine,
C3C3C3, fragment
C8H13NO2) but not in tetraamines
with a central C4 fragment (e.g. spermine or
homospermine; see Fig. 9B). This confirms the identity of
the aminobutylation product of spermidine with canavalmine (Fig.
9A).
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Substrate Kinetics of Deoxyhypusine Synthase and Homospermidine Synthase-- With deoxyhypusine synthase a Km value of 1.3 µM was determined for eIF5A(lys) in the presence of 10 µM spermidine. This Km is well in accordance with values of 0.4 to 3.1 µM reported for the yeast (20), Neurospora (32), and rat and human enzymes (22). Homospermidine synthase shows no activity with the eIF5A precursor protein at various conditions and substrate concentrations (data not shown).
For the two enzymes the apparent Km values were determined in the homospermidine synthase assay for three substrates, i.e. the aminobutyl acceptor putrescine and the aminobutyl donors spermidine and homospermidine. Because spermidine and homospermidine also function as aminobutyl acceptors and thus may compete with putrescine (Table I), apparent Km values were evaluated at three different fixed concentrations of the respective second substrate. The apparent Km values determined for the substrates of the two enzymes are compared in Table II. At low (40 µM) and medium (400 µM) concentrations of the respective non-varied substrates, putrescine shows a slightly higher affinity to homospermidine synthase than to deoxyhypusine synthase, whereas the opposite is true for spermidine. Under the same conditions the two enzymes show almost identical affinities for homospermidine. Generally, with both enzymes the apparent Km values for spermidine and homospermidine rise with increasing concentrations of putrescine as fixed substrates, indicating some competition of putrescine with the two aminobutyl donors. The only distinctive difference between the two enzymes is the about 5-fold higher Km value of putrescine at high constant levels of spermidine (1000 µM) for deoxyhypusine synthase. This implies a somewhat higher affinity of spermidine for the active site in deoxyhypusine synthase than in homospermidine synthase. This is corroborated by the higher efficiency of deoxyhypusine synthase in the formation of canavalmine by aminobutylation of spermidine (Table I). Despite these few quantitative differences the two enzymes display almost identical substrate kinetics. No functional feature becomes noticeable that would qualify homospermidine synthase better adapted in catalyzing homospermidine synthesis than deoxyhypusine synthase. This is particularly true under physiological conditions, i.e. tissue concentrations of spermidine and putrescine of ~30 to 100 µM each (25).
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DISCUSSION |
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The reaction mechanism of deoxyhypusine synthase was mainly studied using the human enzyme. The mechanism is well understood (33-36). The overall reaction occurs in four steps: 1) NAD+-dependent dehydrogenation of spermidine, 2) formation of an enzyme-imine intermediate by transfer of the aminobutyl moiety of dehydrospermidine to the active-site lysine residue (Lys-239 for the human enzyme) (34) and release of diaminopropane as a reaction product, 3) transfer of the same aminobutyl moiety from the enzyme intermediate to a specific lysine residue of the eIF5A precursor protein (Lys-50 for human eIF5A) (37), and 4) by using of the NADH generated in the initial step, reduction of the eIF5A-imine intermediate residue, to form deoxyhypusine. The crystal structure of human deoxyhypusine synthase with bound NAD+ has been obtained (38). The enzyme is a homotetramer consisting of two pairs of identical, tightly associated dimers of 41-kDa subunits. There are four active sites, two active sites in each dimer interface. The catalytic portion of each active site is located in one subunit whereas the NAD binding site is located in the other. Strong evidence indicates that the binding of NAD+ and eIF5A(lys) to the enzyme occurs random, whereas binding of spermidine requires bound NAD+ or NADH (36). Binding of spermidine was not detectable alone or in the presence of eIF5A(lys), suggesting that an NAD+/NADH-induced conformational change is required for the binding of spermidine. An NAD+-dependent binding was also observed with the spermidine analogue N1-guanyl-1,7-diaminoheptane but not with putrescine or spermine. Deoxyhypusine synthases from other sources such as rat (39), yeast (20, 40), and Neurospora (21, 32, 41) are almost identical in their molecular and catalytic properties. This is not surprising, because the amino acid sequences of deoxyhypusine synthases and the eIF5A proteins from various eukaryotic organisms are highly conserved (2). The sequence data of the two plant enzymes fit nicely into this general scheme (12, 13, 19, 42). The Km value of 1.4 µM determined for plant eIF5A(lys) corresponds with those (i.e. 0.4 to 3.1 µM) obtained from non-plant sources.
The specificity of deoxyhypusine synthase toward its substrates NAD+, spermidine, and eIF5A(lys) has been often emphasized as a remarkable feature of the enzyme (29, 36). eIF5A(lys) cannot be replaced by free lysine or synthetic nonapeptides or hexapeptides representing the amino acid sequence surrounding the specific substrate lysine residue of the eIF5A precursor protein (43). To some extent eIF5A(lys) can be truncated at both termini without loss of activity, but the highly conserved sequence region between Phe-30 and Asp-80 is assumed absolutely essential for activity. As far as we are aware no other compounds apart from peptides and amino acids have been tested to replace eIF5A(lys) as substrate. In early studies with partially purified enzymes and 3H-labeled spermidine as substrate, a strong inhibition in the formation of labeled deoxyhypusine was observed in the presence of various diamines and polyamines including homospermidine (44, 45). Later a great number of diamines and polyamine derivatives were tested with the objective to find potential inhibitors of deoxyhypusine synthase (46, 47). In these studies 3-aminopropylcadaverine was found to replace spermidine as substrate (48); with poor efficiency, homodeoxyhypusine instead of deoxyhypusine was formed, indicating the transfer of the aminopentyl residue. The modified eIF5A homologue was inactive in bioassays.
The efficient utilization of putrescine as substrate instead of eIF5A(lys) yielding free homospermidine adds a new characteristic to plant deoxyhypusine synthase. Formation of homospermidine and related amines as products of deoxyhypusine synthase apparently does not seem to play a role under in vivo conditions. However, this side activity of ubiquitous deoxyhypusine synthase may be responsible for the occasional detection of homospermidine in species out of almost all eukaryotic kingdoms, i.e. flowering plants (49-51), mosses (52), ferns (52), algae (53), arthropods (54, 55), invertebrates (56, 57), and vertebrates including humans (58, 59). Sometimes homospermidine was found to be accompanied by the tetraamines canavalmine (28, 51, 52, 57, 59) and homospermine (49, 54, 57), which we identified as minor products of deoxyhypusine synthase. 4-Aminobutylcadaverine, another side product of deoxyhypusine synthase, was also detected in plants (60). A recent survey (61) revealed that of 29 plant species randomly selected from 18 angiosperm families, all were able to produce small amounts of homospermidine.
The results of the protein-protein binding studies and of substrate competition experiments with eIF5A(lys) and putrescine strongly indicate that plant homospermidine synthase (spermidine-specific) (EC 2.5.1.45) can be regarded as a deoxyhypusine synthase (EC 2.5.1.46) that lost the ability to bind and react with eIF5A(lys). Meanwhile this biochemical evidence was directly confirmed by site-specific mutagenesis. Exchanges of amino acids associated with the assumed binding area of the enzyme restore deoxyhypusine synthase activity of homospermidine synthase.2
In S. vernalis, homospermidine synthase occupies an essential role in the biosynthesis of the pyrrolizidine alkaloids, which are important in the plant's defense against herbivory (18). The apparent phylogenetic origin of homospermidine synthase from ubiquitous deoxyhypusine synthase is well supported by the high sequence homology of the two enzymes (13, 19). The results of the present study complete the story; following the basic event of duplication of the dhs gene, the gene copy, i.e. the hss gene, or its encoded protein was subjected to two unpredictable incidents: 1) loss of its essential activity, i.e. aminobutylation of eIF5A(lys), without changing other principle enzymatic properties, including its side activity, i.e. aminobutylation of putrescine; 2) genetic and mechanistic integration into a functionally completely new environment, i.e. pyrrolizidine alkaloid biosynthesis.
In contrast to evolution by gradual modulation of traits, we consider
this sudden and unpredictable evolutionary event creating a new trait
(i.e. function), in analogy to a classical Darwinian term
(62), "molecular evolution by change of function." To our knowledge
there are only a few other examples describing similar events: 1)
putrescine N-methyltransferase, an enzyme committed to
nicotine biosynthesis in tobacco that is suggested as having evolved
from ubiquitous plant spermidine synthase (63); 2) plant tryptophan
synthase, which apparently is a close ancestor of two lyases involved
in the benzoxazinone biosynthesis and the process of indol emission in
maize (64, 65).
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.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.: 49-531-391-5681;
Fax: 49-531-391-8104; E-mail: t.hartmann@tu-bs.de.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M207112200
2 D. Ober and H. J. Hecht, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: eIF5A, eukaryotic initiation factor 5A; HPLC, high performance liquid chromatography; GC-MS, gas chromatography mass spectrometry; MOC, N-methoxycarbonyl; RI, Kovats retention index; Ni-NTA, nickel-nitrilotriacetic acid; DHS, deoxyhypusine synthase; HSS, homospermidine synthase; [M+], molecular ion.
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