©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of Deoxyhypusine Synthase from Neurospora crassa to Homogeneity by Substrate Elution Affinity Chromatography (*)

(Received for publication, July 29, 1994; and in revised form, October 27, 1994)

Yong Tao Kuang Yu Chen (§)

From the Department of Chemistry and Graduate Program in Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08855-0939

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Deoxyhypusine synthase is an NAD-dependent enzyme that catalyzes the formation of deoxyhypusine residue on the eIF-5A precursor by using spermidine as the substrate. Deoxyhypusine synthase bound tightly to 1,12-diaminododecane-agarose and could be eluted selectively by spermidine. This finding enabled us to develop a simple two-column procedure to purify deoxyhypusine synthase from Neurospora crassa to apparent homogeneity. The purified enzyme had a specific activity of 130,000 units/mg of protein, representing a 64,000-fold purification from cell extracts. Size exclusion chromatography indicated that the native enzyme had a molecular mass of 180 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme showed a single band at the 40-kDa position, suggesting that Neurospora deoxyhypusine synthase is a homotetramer. Deoxyhypusine synthase appeared to be hydrophobic and required non-ionic detergent such as Tween 20 to stabilize the activity. Treatment of the enzyme with sulfhydryl reagents resulted in a complete loss of activity. Inclusion of NAD reduced the inactivation rate by manyfold, indicating the presence of -SH groups at or near the active site. Partial amino acid sequences of four peptide fragments that cover about one quarter of the enzyme were obtained for cDNA and genomic cloning work.


INTRODUCTION

Hypusine formation on the eIF-5A (previously named eIF-4D) precursor involves (i) the NAD-dependent cleavage of spermidine and the formation of deoxyhypusine (N-(4-aminobutyl)lysine) (Chen and Dou, 1988) and (ii) hydroxylation of the deoxyhypusine residue (Park et al., 1984). The highly conserved nature of eIF-5A (Park et al., 1993), the responsiveness to growth stimulation and the specificity of hypusine formation (Cooper et al., 1982; Chen, 1983), together with the recognized importance of polyamines in growth regulation (Cohen, 1971; Tabor and Tabor, 1984) suggest that hypusine formation may have an important role in cell physiology. Disruption of the two eIF-5A genes in yeast has been shown to be lethal (Schwelberger et al., 1993). Inhibition of deoxyhypusine synthase (^1)in vivo affects the growth of Chinese hamster ovary cells (Jakus et al., 1993). We have recently developed a rapid assay method for deoxyhypusine synthase (Tao et al., 1994). This assay has allowed us to explore various chromatographic approaches. We found that deoxyhypusine synthase tenaciously binds to 1,12-diaminododecane-agarose and that the enzyme can be selectively released by spermidine. This finding formed the basis for a simple two-column procedure that enabled us to purify deoxyhypusine synthase from Neurospora crassa to apparent homogeneity with good yield.


EXPERIMENTAL PROCEDURES

Materials

Ni(II)-nitrilotriacetic acid-agarose resins were purchased from Qiagen (Chatsworth, CA). [1,8-^3H]Spermidine (17.6 Ci/mmol) was obtained from DuPont NEN. Nitrocellulose membrane (grade BA85) was purchased from Schleicher & Schuell. NAD, N-ethylmaleimide (NEM), (^2)and iodoacetamide (IAM) were obtained from Sigma. Except where noted, all chromatographic matrices and non-ionic detergents were purchased from Sigma. All other chemicals were of standard reagent grade. The polyhistidine-tagged Neurospora 21-kDa eIF-5A precursor, 6xHis-NC21K, was prepared as described (Tao and Chen, 1994).

Growth of Cell Wall-less N. crassa Mutant

The FGSC1118 strain cell wall-less N. crassa mutant cells (Scarborough, 1975) were grown in Vogel's N medium supplemented with 2% (w/v) mannitol, 0.75% yeast extract, and 0.75% nutrient broth for 26-30 h at 30 °C with constant shaking (150 rpm). Cells were harvested by centrifugation (4,000 times g for 20 min).

Enzymatic Assay

The standard reaction mixture, containing 1.2 µg of 6xHis-NC21K, 1 µCi of [^3H]spermidine (final concentration, 3 µM), 1 mM NAD, and enzyme in 0.3 M glycine-NaOH buffer (pH 9.5) in a total volume of 30 µl, was incubated at room temperature for various time periods as indicated. The enzyme reaction was stopped by adding 60 µl of phosphate buffer (0.5 M, pH 6.5) containing 10 mM spermidine. The labeled 6xHis-NC21K was absorbed onto the Ni(II)-nitrilotriacetic acid-agarose and assayed with liquid scintillation spectrometer (Tao et al., 1994).

Other Procedures

SDS-PAGE was carried out as previously described (Dou and Chen, 1990). The native gel electrophoresis was performed using precast mini-PROTEAN II ready gel (4-20% gradient, Bio-Rad) under conditions as described in the instruction manual. Silver staining was carried as described (Blum et al., 1987). Protein amount was determined by the Bradford method(1976) using a Bio-Rad kit.

Purification of Deoxyhypusine Synthase

Wall-less Neurospora cell pellets were stored at -70 °C overnight, thawed, and suspended in buffer A (10 mM phosphate, pH 7.0, 10% glycerol, 1 mM dithiothreitol, and 0.1 mM EDTA) containing 0.2 µM pepstatin A, 0.2 µM leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride. All subsequent steps were carried out on ice or at 4 °C. The mixture was centrifuged at 5,000 rpm for 25 min. The supernatant (260 ml), containing about 5 g of protein, was passed through one layer of cheesecloth and designated as cell extracts. Ammonium sulfate was added to the cell extracts to 30% saturation. The mixture was kept on ice for 1 h and then centrifuged at 10,000 rpm for 30 min. The supernatant was made to 60% saturation with solid ammonium sulfate and was kept at 4 °C for 10 h. The mixture was centrifuged at 10,000 rpm for 30 min. The pellet was dissolved in buffer A for conventional column chromatography using hydroxyapatite column, 1,8-diaminooctane-agarose column, Mono-Q, and Superose-6 columns.

Substrate Elution Affinity Chromatography

The post-ammonium sulfate (60%) fraction (0.6 g of protein) was dissolved in 40 ml of buffer B (10 mM phosphate, pH 7.75, 10% glycerol, 1 mM dithiothreitol, and 0.1 mM EDTA) containing 0.1% Tween 20, mixed with 17.2 ml of 40% PEG8000 (in buffer B), and incubated at 4 °C for 10 h. The mixture was centrifuged at 14,000 rpm for 25 min. The supernatant (53 ml) was mixed again with 40% PEG8000 to a final PEG concentration of 30%. The mixture was kept in ice for 2 h and then centrifuged at 16,000 rpm for 25 min. The precipitate was dissolved in 20 ml of buffer B and designated as post-PEG fraction. The following two columns were then used. (i) In the 1,12-diaminododecyl-agarose (C12) column, the resin (2.0 ml) was washed with water and equilibrated with buffer B containing 0.1% Tween 20. The post-PEG fraction was loaded onto the C12 column (1.6 times 8.5 cm). The column was eluted first with 100 ml of buffer B and then with 50 ml of 1 M NaCl in buffer B, followed by 10 mM spermidine in buffer B (50 ml) containing 1 M NaCl. The fraction eluted by spermidine was concentrated from 50 to 2 ml with Centricon P30. (ii) In the Mono-Q column, the post-C12 sample (2 ml) was diluted in buffer B to about 6 ml and applied onto the Mono-Q column (HR5/5), and the chromatogram was developed with the following program: 0-4 min, 20 mM phosphate buffer (pH 7.75), 0.5 ml/min; 4-24 min, 0.05 M NaCl in buffer B, 0.5 ml/min; 24-84 min, 0.05-0.15 M NaCl in buffer B, 0.5 ml/min. Deoxyhypusine synthase activity always appeared at retention time (68-72 min).

Microsequencing of Deoxyhypusine Synthase Protein

About 4 µg of purified deoxyhypusine synthase was electrophoresed on one-dimensional SDS-polyacrylamide gel. The protein band was transferred from the gel to nitrocellulose membrane (0.45 µ, Schleicher & Schuell) in a 25 mM Tris buffer containing 192 mM glycine and 20% v/v methanol for 15 h at 30 V. The blot was briefly stained with 0.1% Ponceau S (Fluka, Buch, Switzerland) solution in 1% acetic acid and washed in 1% acetic acid. The band was cut with a razor and stored wet at -20 °C in Milli Q water. Sequence determination of trypsin-digested peptide fragments was carried out in the Microchemistry Core Facility at Memorial Sloan-Kettering Cancer Center by a procedure previously described (Tempst et al., 1990).


RESULTS

Initial Purification and Identification of Deoxyhypusine Synthase

The results of an initial purification using conventional approach were summarized in Table 1. The post-Mono-Q fraction gave a specific activity of 39,800 units/mg of protein and represented a 15,000-fold purification from cell extracts. This fraction exhibited two major protein bands on SDS-gel (Fig. 1A, lane2). The use of Superose 6 column resolved these two protein bands (Fig. 1A, lanes3-16). The peak fractions containing the highest amount of deoxyhypusine synthase activity in the Superose 6 chromatogram (Fig. 1B, fractions 7-9) exhibited only the 40-kDa protein band (Fig. 1A, lanes 7-9), suggesting that the 40-kDa protein band was derived from deoxyhypusine synthase.




Figure 1: Purification of deoxyhypusine synthase by Superose 6 column chromatography. Post-Mono-Q fraction in method I was loaded onto a Superose 6 (HR10/30) column. Fractions collected (0.125 ml/fraction at a flow rate of 0.25 ml/min) by Superose 6 column chromatography were used for SDS-PAGE (12% gel) analysis (A) and assay for deoxyhypusine synthase activity (B). A, SDS-PAGE analysis. Each lane contained 10 µl of sample from each fraction. The protein bands were detected by silver staining. Lane1, post-1,8-diaminooctane-agarose fraction; lane2, post-Mono-Q fraction; lanes3-16, samples from fractions 3-16 collected between 61.5 and 68 min. Lanes indicated by M are molecular size markers. B, Superose 6 column chromatogram. Fractions from 3 to 23 were collected for enzyme assay. Samples from fraction 3 to 16 were also analyzed by SDS-PAGE. Sample in fractions 7-9 exhibited a single band at the 40-kDa position on SDS-polyacrylamide gel (A, lanes7-9) and contained the highest enzyme activity. The specific activity in these fractions was about 80 units/µg of protein.



Binding Properties of Deoxyhypusine Synthase

We have examined the binding of the enzyme to 12 different sorbents, including various diaminohydrocarbons. Deoxyhypusine synthase bound tightly to diaminodecane-agarose (C10) and diaminododecane-agarose (C12) at a low salt concentration (data not shown), suggesting that the amino group of C12 may also be involved in the binding with deoxyhypusine synthase. The finding prompted us to examine whether the enzyme can be released specifically from the C12 column by polyamines or diaminohydrocarbons. We found that spermidine (10 mM), but not putrescine or spermine, specifically eluted a 40-kDa protein band as shown by SDS-PAGE. Although diaminodecane or diaminododecane (10 mM each) could release a 40-kDa protein band, they also eluted other major proteins (data not shown).

Purification of Deoxyhypusine Synthase by Affinity Chromatography

The major protein eluted by spermidine from the C12 column appeared to be the deoxyhypusine synthase. Fig. 2A shows the recovery of deoxyhypusine synthase activity in the post-C12 fraction by employing the Mono-Q column. Fig. 2B indicates that the fractions containing high deoxyhypusine synthase activity exhibited a single protein band with an apparent molecular mass of 40 kDa on SDS-polyacrylamide gel. The specific activity of the purified enzyme was about 130 units/µg of protein, representing a 64,000-fold purification from cell extracts. The purified enzyme exhibited a single protein band on a native gel (Fig. 3, lane3). All of the deoxyhypusine synthase activity was found to be associated with this band, indicating that this protein band was indeed deoxyhypusine synthase. We therefore concluded that a combination of two columns, namely, the C12 and Mono-Q, has led to the purification of deoxyhypusine synthase to an apparent homogeneity. The results of a representative purification are summarized in Table 2.


Figure 2: A, purification of deoxyhypusine synthase by C12 and Mono-Q column chromatography. The post-C12 fraction eluted by spermidine was concentrated to 2 ml (40 µg/ml) and then loaded onto the Mono-Q column. The chromatogram was developed by a program as described under ``Experimental Procedures.'' The fractions collected between 68 and 72 min contained pure deoxyhypusine synthase. B, SDS-PAGE analysis of purified Neurospora deoxyhypusine synthase. LaneM, protein standard (68, 45, and 30 kDa as indicated); lane1, 1 µg of post-Mono-Q fraction; lane2, 4 µg of post-Mono-Q fraction; lane3, 0.5 µg of ovalbumin (OV); lane 4, 2 µg of ovalbumin; lane5, 4 µg of ovalbumin. The gel was stained by 0.1% Ponceau S.




Figure 3: Native gel electrophoresis of pure deoxyhypusine synthase. The native gel was treated by silver staining. Lane1, protein standard (240, 67, and 45 kDa as indicated); lane2, 240-kDa protein; lane3, post-Mono-Q fraction (0.5 µg of protein). An identical post-Mono-Q sample was run on another lane for enzymatic assay. The gellane was sliced (4 mm/slice) and minced in the assay buffer. The enzyme activity in each slice was measured using SDS-PAGE and fluorography as described before (Dou and Chen, 1990).





Estimate of the Molecular Mass of the Native Enzyme

Size exclusion chromatography using Superose 6 HR10/30 indicated that the native deoxyhypusine synthase had an apparent molecular mass of 180 kDa (data not shown). However, SDS-PAGE analysis of purified deoxyhypusine synthase revealed a single band at 40 kDa (Fig. 2B). These results suggest that Neurospora deoxyhypusine synthase is likely a homotetramer. Since a single protein was found to be associated with deoxyhypusine synthase activity, we concluded that deoxyhypusine synthase is a single multifunctional enzyme that is capable of catalyzing both the spermidine dehydrogenation and the transfer of aminobutyl group to the eIF-5A precursor protein.

Partial Amino Acid Sequences of Deoxyhypusine Synthase

Four internal peptide fragments derived from the Neurospora deoxyhypusine synthase though in situ trypsin digestion were sequenced. The partial amino acid sequences of these peptides are HVSLIVTTAGGIEEDSIK, NGAESAVYINTAQEFD, NDIPVFCPALTDGWLGDMLK, and IGNLVVPNSNYCAFEDWVVPI. If deoxyhypusine synthase subunit has a molecular mass of 40 kDa, the 75 amino acid residues in these peptides represent approximately 25% total sequence of the enzyme. The GenBank search indicated that these sequences did not share any homology with other known proteins.

Hydrophobicity and Stability of Deoxyhypusine Synthase

Deoxyhypusine synthase bound tightly to phenyl-Sepharose column in the presence of high salt concentration (1 M Na(2)SO(3) in buffer B), suggesting the presence of hydrophobic patches at the surface of the enzyme (data not shown). Freeze-thawing of pure or partially purified enzyme, even in the presence of 10% glycerol, caused a rapid loss of enzyme activity. Non-ionic detergent such as Tween 20 was effective in maintaining the enzyme activity against multiple freeze-thawing (data not shown). The hydrophobic nature and the multisubunit structure of the protein may contribute to the instability of the enzyme.

Protection Against the Inhibition of Sulfhydryl Reagents by NAD

Fig. 4illustrates the inhibitory effects of both NEM and IAM on purified deoxyhypusine synthase. The results indicate a direct inactivation of the enzyme by NEM and IAM. Fig. 4also shows that the inactivation could be partially blocked by pre-incubation of the enzyme with NAD, suggesting the presence of cysteine residues at or near NAD binding site. Alternatively, NAD may induce global conformational change that renders exposed cysteine group(s) cryptic.


Figure 4: Protection of deoxyhypusine synthase from the inhibition by sulfhydryl reagents. Deoxyhypusine synthase (Post-Mono-Q fraction from method I) was mixed without or with different concentrations of NAD for 5 min, followed by an incubation without (control) or with 10 mM of IAM or 1 mM of NEM for another 30 min at room temperature. Enzyme assays were then carried out as described under ``Experimental Procedures.''




DISCUSSION

We described here for the first time the purification of deoxyhypusine synthase from N. crassa to apparent homogeneity as judged by SDS-PAGE. The initial purification using the conventional chromatographic approach resulted in a 31,000-fold purification but with only 0.25% yield (Table 1). In contrast, the two-columns method is rapid and simple, and the recovery is good (Table 2). From the degree of purification ( Table 1and Table 2), we estimated the abundance of deoxyhypusine synthase in wall-less Neurospora mutant cells to be about 0.001% or less. The abundance of 21-kDa eIF-5A, the modified substrate protein, in wall-less mutants was estimated to be about 0.1% by immunostaining. (^3)Gel filtration and SDS-PAGE analysis of purified deoxyhypusine synthase suggest that the enzyme is a homotetrameric protein consisting of four identical subunits.

The binding affinity of deoxyhypusine synthase to diaminododecane resembles that of putrescine oxidase from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens (Okada et al., 1979), suggesting that there may exist certain structural similarities among these enzymes. Whether the hydrophobic nature of the enzyme may affect its stability is not clear. We noticed, however, that during purification the enzyme became less stable as the degree of purification increased. The finding that non-ionic detergent such as Tween 20 stabilizes the enzyme activity may suggest the intracellular environment of the enzyme is also hydrophobic in nature. It is noteworthy that spermidine dehydrogenase in Citrobacter freundii has been found in membranous fractions (Hisano et al., 1992). The finding that deoxyhypusine synthase is sensitive to sulfhydryl reagents (Fig. 4) indicates that cysteine residues are required for enzymatic action. It also suggests the possibility that the enzyme can be subjected to redox regulation. The finding that NAD can protect the enzyme from the inhibition strongly suggests that the cofactor NAD may cause some conformational change of the enzyme. This is consistent with the finding that the enzyme binds to its substrate protein only in the presence of NAD (Tao and Chen, 1994).


FOOTNOTES

*
This study was supported in part by United States Public Health Service Grant RO1 CA49695 awarded by NCI, National Institutes of Health, and a grant from The Charles and Johanna Busch Memorial Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Chemistry, Rutgers University, P. O. Box 939, Piscataway, NJ 08855-0939. Tel.: 908-445-3739; Fax: 908-445-5312; KYCHEN{at}mbcl.rutgers.edu.

(^1)
According to the conventions of the Enzyme Nomenclature Commission, the enzyme should be named spermidine dehydrogenase (NAD, 1-deoxyhypusyl-forming and aminobutyl-transferring). In accordance with previous publications, the trivial name ``deoxyhypusine synthase'' is used in this paper.

(^2)
The abbreviations used are: NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; C12, 1,12-diaminododecane-agarose; PEG, polyethylene glycol; IAM, iodoacetamide; 6xHis-NC21K, polyhistidine-tagged Neurospora 21-kDa eIF-5A precursor protein.

(^3)
Y. Tao and K. Y. Chen, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Dr. John Lenard (Robert Wood Johnson Medical School, UMDNJ, NJ) for giving us the wall-less N. crassa mutants. We thank Dr. Scott Geromanos, Sloan-Kettering Institute for carrying out the microsequencing work.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.