(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 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.
Hypusine formation on the eIF-5A precursor involves (i)
NAD-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).
A general
polymerase chain reaction (PCR) ()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.
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 1.3-kb fragment (Fig. 2D, lane2). This 1.3-kb
fragment was subcloned into pBluescript and sequenced.
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 () 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.
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 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.
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).