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(Received for publication, May 11, 1995; and in revised form, July 31, 1995) From the
Deoxyhypusine synthase catalyzes the formation of deoxyhypusine
residue on the eIF-5A precursor using spermidine as the substrate. We
have purified deoxyhypusine synthase from Neurospora crassa to
apparent homogeneity (Tao, Y., and Chen, K. Y.(1995) J. Biol. Chem. 270, 383-386). We have now cloned and characterized the
deoxyhypusine synthase cDNA using a reverse genetic approach.
Conceptual translation of the nucleotide sequence of the cloned
1258-base pair cDNA revealed an open reading frame containing 353 amino
acids with a predicted M
Hypusine formation on the eIF-5A precursor involves (i)
NAD A general
polymerase chain reaction (PCR) (
Figure 1:
A, renaturation of deoxyhypusine
synthase from SDS-PAGE. Post-C12 (1,12-diaminododecane-agarose) column
enzyme preparation was separated by SDS-PAGE. Gel slices corresponding
to the 110-, 55-, 40-, and 29-kDa protein bands were excised and
extracted by 1 ml of buffer for 2 h at 4 °C. The extraction
solution were subjected to three cycles of concentration-dilution.
Deoxyhypusine synthase activity was detected by SDS-PAGE and
fluorography. Lane1, input enzyme; lane2, gel slice at 29 kDa; lane3, gel
slice at 40 kDa; lane4, gel slice at 55 kDa; lane5, gel slice at 110 kDa. The arrow indicates the position of radiolabeled 6xHis-NC21K. B,
design of specific degenerate primers. Amino acid sequences of three
tryptic peptides, T51, T53, and T35, were shown. The arrows labeled P1-P6 represent the amino acid residues
used for designing specific, degenerate
primers.
Figure 2:
A,
the 3`- and 5`-end cDNA cloning strategy. For 3` end, a specific sense
primer (P3 in this case) was used with universal primer, MR, to amplify
the cDNA library under the touchdown PCR conditions. The PCR product
was used in the second PCR as the template to be amplified by the
second sense primer (P1 or P5 in this case). For 5`-end, a
gene-specific antisense primer, P10, was first used with universal
primer to amplify the cDNA library. The PCR product was used in the
second PCR as the template to be amplified by a second gene-specific
antisense primer P8 and universal primer (MR or MF). B, PCR
products from the 3`-end cDNA cloning. PCR products were analyzed by
agarose (1%) gel electrophoresis. Each product was denoted by the
primers used for amplification. Thus R13 represents PCR product
resulted from two runs of PCR using (MR + P1) in the first run and
(MR + P3) in the second run. Lane1, R13; lane2, R15; lane3, R31; lane4, R35; lane5, R51; lane6, R53; lane7, Life Technologies 1-kb
DNA marker. C, PCR products from the 5`-end cDNA cloning. Lane1, 1-kb DNA marker; lane2, 10
µl of final PCR products, R108. D, ligase-free PCR
recombination. Lane1, control, no DNA template; lane2, PCR product from ligase-free recombination; lane3, PCR product from 5`-end cDNA by amplified by
P8 and MR; lane4, PCR product from 3`-end cDNA
amplified by P3 and MR.
The sequence information obtained for
the 3`-end cDNA of deoxyhypusine synthase enabled us to design two
gene-specific antisense primers, P8 and P10, for cloning the 5`-end
cDNA of the enzyme. Fig. 2C shows the agarose gel
analysis of the PCR products after the second PCR run. The most
prominent band has the size of about 700 bp (lane2).
Sequence analysis revealed that the 700-bp fragment contained an ORF
with sequences matching tryptic peptides T53 and T4. The 3`-end and
5`-end cDNA were amplified (Fig. 2D, lanes3 and 4) and combined by using two universal
primers in a ligase-free PCR reaction. The combination of the 700-bp
5`-end cDNA and 600-bp 3`-end cDNA produced a
Figure 3:
Nucleotide sequence and predicted amino
acid sequence (single-letter amino acid code) of Neurospora deoxyhypusine synthase. The ORF defined by
assigning the initiation codon ATG at position 37, is in frame with
amino acid sequences of four tryptic peptides previously determined (underlined). The translation stop codon (TGA) is shown with
an asterisk.
Figure 4:
Alignment of the amino acid residues of
human (partial), yeast, and Neurospora deoxyhypusine synthase.
The amino acid sequence is shown in single-letter
code. Gaps (
We also found a short human expressed sequence tag
(Z25337(1993)) that bears considerable homology to Neurospora deoxyhypusine synthase cDNA. The amino acids encoded by this
312-bp human sequence covers from residues 93 to 196 (Fig. 4).
It can be noted that a striking homology exists in the amino acid
sequence extending from 101 to 196 for human, yeast, and Neurospora polypeptides, with amino acid similarity as high as 85%,
suggesting that deoxyhypusine synthase is a highly conserved enzyme.
The identification of this expressed sequence tag as a partial human
deoxyhypusine synthase sequence proves invaluable in our cloning of two
full-length human cDNAs for deoxyhypusine synthase. ( The
hydropathy profiles of Neurospora and yeast enzyme are nearly
superimposable (data not shown), consistent with their high sequence
homology. In the case of Neurospora deoxyhypusine synthase,
both the N and C termini of this enzyme appear to be highly
hydrophobic. Whether this may be related to the hydrophobic
chromatographic behavior of the Neurospora enzyme remains to
be examined.
Figure 5:
Expression of pQDS in M15 E. coli.A, protein pattern on a SDS-PAGE. Cell lysates were
prepared as described (Tao and Chen, 1994). Lane1,
protein standard; lane2, lysate (2 µl) from the
pQE60-transfected cells; lane3, lysate (2 µl)
from pQDS-transfected cells. B, deoxyhypusine synthase
activity of the recombinant protein. The radiolabeled 6xHis-NC21K
substrate protein was detected by autoradiography after SDS-PAGE. Lane1, lysates from pQDS transformant (1 µl); lane2, lysates from pQDS transformant (5 µl); lane3, lysates from pQE60 transformant (1 µl); lane4, lysates from pQE60 transformant (5
µl).
The demonstration that the 40-kDa polypeptide exhibited
deoxyhypusine synthase activity after renaturation (Fig. 1) and
the functional expression of the cloned deoxyhypusine synthase cDNA in
bacteria (Fig. 5) strongly suggest that the 40-kDa subunit alone
constitutes the active tetrameric enzyme that catalyzes both oxidative
cleavage of spermidine and subsequent transfer of the aminobutyl moiety
to eIF-5A precursor. The cloned 1258-bp cDNA contains one ORF that
covers the entire amino acid sequence of deoxyhypusine synthase (Fig. 3). This conclusion is supported by the following
evidence: (i) the molecular mass of the predicted amino acid sequence
corresponds to that determined by SDS-PAGE; (ii) the Kozak consensus
sequence precedes the initiation codon; (iii) the predicted amino acid
sequence contains four tryptic peptide fragments that we previously
sequenced; (iv) the predicted amino acid sequences from different
species share high homology (Fig. 4); and (v) the recombinant
protein exhibits deoxyhypusine synthase activity (Fig. 5). Based on the amino acid sequence alignment (Fig. 4), it is
likely that we have also obtained the complete sequence for yeast
deoxyhypusine synthase and about one third of human sequence. The
homology between Neurospora, yeast and human (partial) protein
is striking. Of particular note are two stretches of amino acids, one
spans from residue 101 to 196, and the other from 220 to 352 (yeast
numbering). Consistent with the high degree of homology, the hydropathy
profiles of both Neurospora and yeast proteins are similar
(data not shown). Neurospora deoxyhypusine synthase has been
shown to bind tightly to phenyl-Sepharose column in the presence of
high salt buffer, suggesting the presence of hydrophobic patches at the
enzyme surface (Tao and Chen, 1995). These patches cannot be clearly
identified based on hydropathy plot. Deoxyhypusine synthase is a
bifunctional enzyme that utilizes NAD
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U22400[GenBank]. Note Added in Proof-While this
manuscript was under review, two papers describing the identification
of yeast deoxyhypusine synthase cDNA were published (Klier, H., Csonga,
R., Steinkasserer, A., Wohl, T., Lottspeich, F., and Eder, J.(1995) FEBS. Lett.364, 207-210 and Kang, K. R., Wolff,
E. C., Park, M. H., Folk, J. E., and Chung, S. I.(1995) J. Biol.
Chem.270, 18408-18412).
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 23984-23987
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of 38,985. The
deoxyhypusine synthase cDNA was subcloned into the expression vector
pQE60 to produce a 40,000-dalton recombinant protein on SDS-PAGE which
exhibited deoxyhypusine synthase activity. A GenBank search showed that
the Neurospora deoxyhypusine synthase cDNA possessed
significant sequence homology to a previously uncharacterized yeast
sequence (accession number U00061(1994)). The yeast sequence encodes a
protein of 387 amino acids that shows 69% of total amino acid identity
and 80% of total amino acid similarity to the Neurospora enzyme. Sequence alignment and hydropathy analysis suggest that
the yeast sequence represents deoxyhypusine synthase.
-dependent oxidative cleavage of spermidine, (ii)
transfer of the aminobutyl moiety derived from spermidine to eIF-5A
precursor to form deoxyhypusine (N
-(4-aminobutyl)lysine) residue, and (iii)
hydroxylation of the deoxyhypusine residue (Park et al., 1984;
Chen and Dou, 1988; Park and Wolff, 1988). Deoxyhypusine synthase
catalyzes the first two steps in hypusine formation. Disruption of the
two eIF-5A genes in yeast has been shown to be lethal (Schnier et
al., 1991). Inhibition of deoxyhypusine synthase by N
-guanyl-1,7-diaminoheptane causes growth arrest
of Chinese hamster ovary cells (Jakus et al., 1993) and
differentiation of mouse neuroblastoma cells. ()Deoxyhypusine synthase has recently been purified from Neurospora crassa and appears to be a homotetramer with a
subunit size of 40,000 daltons (Tao and Chen, 1995).
)approach has been outlined
using a single-sided specific primer in conjunction with nonspecific
primers targeted either to the 3` poly(A) region or to
an enzymatically synthesized tail at 5`-end that permits amplifications
of the regions upstream and downstream of the core sequence (Frohman et al., 1988; Ohara et al., 1989). Using partial
amino acid sequence information obtained from Neurospora deoxyhypusine synthase (Tao and Chen, 1995), we have adopted a
simple PCR strategy to clone Neurospora deoxyhypusine synthase
cDNA. Here we report the molecular cloning and functional expression of
recombinant deoxyhypusine synthase in vitro and in
vivo. In addition, we have also identified a hitherto
uncharacterized yeast sequence in GenBank that most likely represents
the yeast deoxyhypusine synthase cDNA.
Materials
Escherichia coli strains,
plasmids, and chemicals were the same as described previously (Tao and
Chen, 1994).Renaturation of Deoxyhypusine Synthase after SDS-PAGE
Analysis
Partially purified deoxyhypusine synthase was separated
by SDS-PAGE at 4 °C. Gel slices were rinsed with extraction buffer
(20 mM phosphate buffer, pH 7.75, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol, and 0.1% Tween
20) and homogenized. The mixture was spun in a centrifuge, and the
supernatant was concentrated using Centricon 30 for 1 h at 4 °C.
The concentration step was repeated three times. The concentrated
sample was used for the enzyme assay as described (Dou and Chen, 1988). Design and Synthesis of Oligonucleotide Primers for PCR
Reaction
Six degenerate primers were synthesized based on the
partial amino acid sequences of the deoxyhypusine synthase: P1
(192-fold degeneracy), 5`-dAACGACATHCCNGTNTTTTGYCC; P2 (256-fold
degeneracy), 5`-dAACATATCNCCNARCCAACCRTC; P3 (32-fold degeneracy),
5`-dCAACTACTGYGCNTTYGARGA; P4 (128-fold degeneracy),
5`-dCCAGTCYTCRAANGCRCARTARTT; P5 (128-fold degeneracy),
5`-dTACATCAAYACNGCNCARGARTT; P6 (384-fold degeneracy),
5`-dGTCGAAYTCYTGNGCNGTRTTDAT. (H = T + C + A, N
= T + C + A + G, Y = T + C, R
= A + G, and D = A + G + T). Other primers
used were: P8, 5`-dGTGGCCTAGACTGCCGTCGGTGAC-3`; P10, 5`-dGGCCTTG
AACGTATGGAAATACAG-3`; P12, 5`-dACTTGGAATCTGGTTGTCCGCCAT-3`; universal
primers MF 5`-dGCCAGGGTTTTCCCAGTCACGA-3` and MR
5`-dGAGCGGATAACAATTTCACACAGG-3`.Amplification of 3`-End and 5`-End Deoxyhypusine Synthase
cDNA Fragment by PCR
The reaction mixture in the first PCR run
contained 0.5 µl of cDNA library, as well as a degenerate sense
primer and a universal primer (0.2 µM each). The first run
PCR product was then used as template and amplified by another sense
degenerate primer and a universal primer in the second PCR run. In both
runs, the ``touchdown'' protocol (Don et al., 1991)
was followed. The amplified 3`-end cDNA fragments were subcloned into a SmaI-EcoRI-digested pBluescript KS vector for
sequence determination. Two gene-specific antisense primers, P8 and
P10, based on the sequence of the 3`-end cDNA were used to obtain the
5`-end cDNA fragment. Primer P10 and universal primer, MR, were used in
the first run to amplify the Neurospora cDNA library. The
first run PCR product (0.1 µl) was then used as template for the
second run amplification by primers P8 and MR under the same PCR
condition. Clones containing either the 5`-end cDNA or 3`-end cDNA
inserts were amplified by PCR separately. The amplified 5`-end and
3`-end cDNA fragments were gel-purified, mixed together, and amplified
by a pair of universal primers (Shuldiner et al., 1990).
Plasmids were sequenced by the dideoxynucleodtide chain-termination
method (Sanger et al., 1977). The computer analysis of DNA and
deduced protein sequences were carried out by the program of the GCG
package version 7.3.1 and MacVector 4.5 from IBI/Kodak.Expression of Deoxyhypusine Synthase in TNT(TM)
Reticulocyte Lysate and in E. coli
The pBluescript KS(II)
constructs containing cDNA inserts were used as templates in
TNT(TM)-coupled transcription-translation system (Promega, Madison,
WI). T3 RNA polymerase was used to drive the synthesis of deoxyhypusine
synthase mRNA. About 1 µg of circular plasmid DNA was added into a
50-µl TNT(TM) rabbit reticulocyte lysate reaction mixture, which
includes all components for transcription and translation. Other
details were described in the manufacturer's technical bulletin.
PCR was used to generate the NcoI site before the first ATG of
deoxyhypusine synthase. The PCR product was sublcloned into pQE60
vector (Qiagen, Chatsworth, CA). The construct, designated as pQDS was
used to transform E. coli M15 strain (Tao et al.,
1994).
Renaturation of Deoxyhypusine Synthase from
SDS-PAGE
To examine whether the 40-kDa polypeptide alone is
sufficient to account for the deoxyhypusine synthase activity, we have
tested the activity of the 40-kDa polypeptide after renaturation from
SDS-polyacrylamide gel. Fig. 1A shows that only the
40-kDa band exhibited deoxyhypusine synthase activity after
renaturation. The overall recovery of the original enzyme activity was
about 10%. Other protein bands recovered from gel slices did not have
any effect on the renatured deoxyhypusine synthase, suggesting that the
40-kDa polypeptide represents the only subunit for Neurospora deoxyhypusine synthase. Based on the partial amino acid sequence
information, we have designed three pairs of degenerate primers,
P1-P6, to gain entry into the Neurospora cDNA library (Fig. 1B).
PCR Cloning of 3`-End and 5`-End cDNA Fragments and
Ligase Free Recombination
Fig. 2A illustrates
the PCR strategy for cloning the 3`-end and 5`-end cDNA fragments. The
use of different combination of nested primers not only significantly
enhanced the specificity of amplification, they also provided
information on the order of these sequences. Fig. 2B shows that for 3`-end cDNA amplification, only R31 and R35 gave
prominent DNA bands, indicating the order of primers is P3-P1 and
P3-P5. The most prominent bands in R31 and R35 had the size of
about 650 and 450 bp, respectively. Sequence analysis demonstrated that
these 650 bp contained one open reading frame (ORF) and a poly(A) tail.
The translation of this ORF matched the amino acid sequences of peptide
T51 and T35, confirming that we have obtained the 3`-end cDNA fragment
for deoxyhypusine synthase.
1.3-kb fragment (Fig. 2D, lane2). This 1.3-kb
fragment was subcloned into pBluescript and sequenced.
Sequence Analysis of Deoxyhypusine Synthase cDNA
Fig. 3shows the entire nucleotide sequence of the cDNA,
including the 5`- and 3`-untranslated regions of 36 and 160 bp,
respectively. The cDNA contained one ORF, encoding a protein of 353
amino acids with molecular mass of 38,985. All four tryptic peptide
fragments previously sequenced can be identified within this ORF. The
predicted amino acid sequence of Neurospora deoxyhypusine
synthase is not related to any other known proteins in the GenBank and
EMBL data bases. However, we found that the Neurospora deoxyhypusine synthase cDNA shows a high homology to a hitherto
uncharacterized yeast sequence (YHRO68w, accession number U00061(1994),
located on chromosome VIII). The yeast sequence contains one ORF
encoding a protein of 387 amino acids. Fig. 4shows the
alignment of this 387-amino acid sequence with that of Neurospora deoxyhypusine synthase. The overall amino acid sequence identity
between the Neurospora deoxyhypusine synthase and the putative
yeast enzyme is 69%, and the overall amino acid sequence similarity is
80%. The degree of amino acid identity is not distributed equally,
being highest in the middle of the polypeptide but not at the N
terminus. For example, a 132-amino acid sequence extending from
residues 220 to 352 (yeast numbering) shows 81% identity and 94%
similarity between these two polypeptides. Such high homology suggests
that the U00061 sequence represents the yeast deoxyhypusine synthase
cDNA.
) were introduced for maximal alignment of the
polypeptides. Numbering of amino acids begins with the first amino acid
residue of predicted yeast deoxyhypusine synthase
sequence.
)Functional Expression of Recombinant Deoxyhypusine
Synthase in Vitro and in Vivo
We first tested functional
expression of recombinant protein in vitro. A significant
incorporation of radioactive spermidine into 6xHis-NC21K was detected
(1,000 cpm over the control for 1 µg of plasmid) when the
reticulocyte lysate was programmed with plasmid containing
deoxyhypusine synthase cDNA insert (data not shown). We then examined
the enzyme activity of recombinant protein expressed in E.
coli. Fig. 5A shows the
isopropyl-1-thio-
-D-galactopyranoside induction-induced
expression of the 40-kDa protein in transfected M15 cells (Fig. 5A, lane3versuslane2). The lysates from pQDS-transfected cells
were directly used to test for the presence of deoxyhypusine synthase
activity. Fig. 5B shows that deoxyhypusine synthase
activity could be detected in cell lysates transfected by pQDS after
isopropyl-1-thio--D-galactopyranoside induction (Fig. 5B, lanes1 and 2versuslanes3 and 4). Taken
together, these results confirm that the cloned 1258-bp cDNA represents
the gene for deoxyhypusine synthase from N. crassa.
as co-factor
(Chen and Dou, 1988). Surprisingly, the deduced amino acid sequence of Neurospora or yeast deoxyhypusine synthase fails to share any
similarities with other known dehydrogenases. We also failed to detect
the nucleotide binding domain,
Gly-X-Gly-X-X-Gly (Rossmann et al.,
1974) in either Neurospora or yeast sequence. Nevertheless,
the binding sites for NAD
and for the two substrates,
spermidine and eIF-5A precursor, are likely to be located in the most
conserved region in the protein. We have shown that the binding of
eIF-5A precursor to deoxyhypusine synthase occurs only in the presence
of NAD
(Tao and Chen, 1994). We have also shown that
the presence of NAD
protects the enzyme from the
inhibitory action of sulfhydryl reagents (Tao and Chen, 1995). These
results prompt us to speculate that the two cysteine residues
(positions 145 and 180), located within the most conserved region in
deoxyhypusine synthase (Fig. 4), may be near or within the
active site. The tetrameric structure of deoxyhypusine synthase also
raises the possibility that the four subunits of the enzyme may
generate two pockets for spermidine binding when they are assembled.
Further studies are needed to elucidate the enzyme structure and its
interaction with co-factor and substrates. The availability of
deoxyhypusine synthase cDNA should prove invaluable in achieving these
goals.
)))
We are grateful to Dr. John Lenard, Robert Wood
Johnson Medical School, UMDNJ, NJ, for giving us the N. crassa cDNA library. We also thank Dr. Zong Ping Chen and Andrea J. Kwon
for graphic work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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