Expression purification and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum

Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate

dehydrogenase from Clostridium acetobutylicum

Abdelghani Iddar, ^a Federico Valverde, ^b Aurelio Serrano, ^b,1 and Abdelaziz Soukri ^a,*

a

Laboratoire de BBCM, D eepartement de Biologie, Facult ee des Sciences A€ ıın-Chock, Universit ee Hassan-II. Km 8 route d’El Jadida, B.P. 5366 M^ a aarif, Casablanca, Morocco

b

Instituto de Bioqu ıımica Vegetal y Fotos ııntesis (CSIC-Universidad de Sevilla), Centro de Investigaciones Cient ııficas Isla de la Cartuja,

Am eerico Vespucio s/n, Sevilla 41092, Spain

Received 11 January 2002, and in revised form 28 February 2002

Abstract

Clostridium acetobutylicum gapN was cloned and expressed in Escherichia coli BL-21. The IPTG-induced nonphosphorylating NADP-dependent GAPDH (GAPN) has been purified about 34-fold from E. coli cells and its physical and kinetic properties were investigated. The purification method consisted of a rapid and straightforward procedure involving anion-exchange and hy- droxyapatite chromatographies. The purified protein is an homotetrameric of 204 kDa exhibiting absolute specificity for NADP.

Chromatofocusing analysis showed the presence of only one acidic GAPN isoform with an acid isoelectric point of 4.2. The op- timum pH of purified enzyme was 8.2. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 65 °C with activation energy of 18 KJ mol

. The apparent K

values for NADP and

-glyceraldehyde-3-phophate (

-G3P) or

-G3P were estimated to be 0:200 0:05 and 0:545 0:1 mM, respectively. No inhibition was observed with L-D3P. The V

of the purified protein was estimated to be 78:8 U mg

. The Cl. acetobutylicum GAPN was markedly inhibited by sulfhydryl- modifying reagent iodoacetamide, these results suggest the participation of essential sulfhydryl groups in the catalytic activity.

Ó 2002 Elsevier Science (USA). All rights reserved.

Keywords: Clostridium acetobutylicum; Nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN); Expression;

Purification

Glyceraldehyde-3-phosphate dehydrogenase (GAP- DH) is an enzyme involved in central pathways of carbon metabolism [1]. Three distinct GAPDH proteins with different subcellular localization and performing various roles have been found. (i) A typical NAD-dependent glycolytic enzyme (EC. 1.2.1.12) found in all organisms so far studied and located in the cytoplasm, that plays a pivotal role in the Embden–Meyerhoff pathway not only in glycolysis but also in glyconeogenesis [1]. (ii) The NADP-dependent GAPDH (EC. 1.2.1.13), a key com- ponent of the reductive pentose-phosphate (RPP) cycle, which is located in the chloroplast stroma and the

cyanobacterial cytoplasm and is involved in photosyn- thetic CO

assimilation [2,3]. These two enzymes catalyze the oxidation of glyceraldehyde-3-phosphate (G3P) into diphosphoglyceric acid using the orthophosphate anion (PO

) as a substrate in their catalytic reactions. (iii) The cytosolic non-phosphorylating NADP-dependent GAPDH (GAPN) (EC. 1.2.1.9), also named Pi-indepen- dent G3P:NADP oxidoreductase, then has been pro- posed to metabolism trioses exported from chloroplast.

This enzyme catalyzes an irreversible oxidation of G3P to 3-phosphoglycerate [4,5].

The GAPN has originally been reported to be only present in green eukaryotes [4]. However, early reports of GAPN activity in Streptococcus mutans and S. sali- varius [6] were confirmed by molecular data [7]. Fur- thermore an archaeal nonphosphorylating GAPDH has been biochemically described from Thermoproteus tenax

www.academicpress.com

*

Corresponding author. Fax: +212-22-23-06-74.

E-mail addresses: aurelio@cica.es (A. Serrano), a_soukri@hotmail.

com (A. Soukri).

1

Also corresponding author. Fax: +34-4460065.

1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.

PII: S 1 0 4 6 - 5 9 2 8 ( 0 2 ) 0 0 0 3 2 - 3

[8]. So far the GAPN has not been found in fungi and animals.

The GapN gene encoding for maize and microalga Chlorela fusca GAPN was already cloned [9]. The GAPN of higher plants [10,11] and bacteria [7] se- quenced so far consist of a subunit of about 490 amino acids. The active enzyme in plants is homo-tetramer of about 190 kDa as determined from beet, Chlamydo- monas reinhardtii and Hevea brasiliensis [12–14].

The cloning of the GAPN revealed that is a member of the large aldehyde dehydrogenase (ALDH) superfamily.

The GAPN differs from GAPDHs both in primary structure and molecular mass [10,13,15,16]. The super- family is divided into several branches each representing a gene family [10,11]. The bacterial GAPN shows about 30–

50% amino acid identity to the eukaryotic enzyme [11].

Up to now, to our knowledge, no data describing the presence and characterization of G3P:NADP oxydore- ductase Pi-independent in Clostridium acetobutylicum were reported. In this work, we have cloned the Cl.

acetobutylicum gapN in a PET-3a expression systems, so the functional GAPN could be expressed in Escherichia coli BL-21. The recombinant GAPN was purified and characterized.

Materials and methods

Chemical and plasmids

D

-glyceraldehyde-3-phosphate (

-G3P) and

-gly- ceraldehyde-3-phosphate (

-G3P) were prepared from monobarium salts of the diethyl acetal (Sigma); all other chemicals (analysis grade) were from Fluka or Merck.

Cloning of PCR product and expression were performed using the plasmids pGEM-T (Promega) and PET-3a (Stratagene), respectively.

Organisms and growth conditions

Clostridium acetobutylicum ATCC 824 strain was cultivated in typticase-yeast-extract-glucose (TYA) broth [17] at 37 °C in an anaerobic chamber in nitrogen (N

) atmosphere.

Escherichia coli K-12, DH5a, BL-21 strains, and complemented BL-21 clones were cultivated at 37 °C in Luria–Broth (LB) [18]. Either solid (plus 1.5%, w/v, Difco-Bactoagar) or liquid cultures were used. When necessary, ampicilline (Amp) and isopropyl-b-thioga- lactopyranoside (IPTG) were added at concentrations of 100 lg ml

and 1 mg ml

, respectively.

Enzyme assay

The GAPN activity was measured as described in [9].

The reaction was started by adding the enzyme to the

assay mixture containing 50 mM tricine buffer (pH 8.5), 3 mM 2-mercaptoethanol, 1 mM NADP, and 1 mM

D D L

D

-G3P, or

-G3P at 25 °C. The absorbance variation at 340 nm was followed. The phosphorylating NAD- dependent GAPDH activity was measured using the same procedure with NAD (1 mM) and inorganic phosphate PO

or arsenate AsO

(10 mM) in the reaction medium.

Kinetic constants were calculated from initial rates.

For the determination of kinetic parameters, the con- centrations of the respective fixed substrate for the re- action were 1 mM NADP or 0.2 mM

-G3P or

-G3P.

K

and V

were determined from lineweaver-Burk double-reciprocal plots. The optimal pH was estimated in the range from 5 to 13 using different buffers (acetate, imidazole, Tris, and carbonate/bicarbonate) adjusted to the same ionic strength as the standard mixture. Ther- mal inactivation experiments were carried out by en- zyme incubation in 20 mM Tris–HCl and 2 mM EDTA (pH 7.5) over temperature range from 15 to 95 °C. After 10 min of incubation, aliquots were cooled in an ice bath and the residual activity was determined as described above. Thermal activation was carried out by measuring the activity in 50 mM tricine buffer at temperature range from 15 to 80 °C. One unit of enzyme is defined as the amount that catalyzes the formation of 1 lmol NADPH per minute under the specified conditions.

Cloning strategy and DNA manipulation

Chromosomal DNA was isolated from Cl. acetobu-

tylicum strain, using the WIZARD Kit (Promega). A

PCR amplification was carried out 50 °C with two

complementary oligonucleotides (forward, 5

-GTGGT

GTACCATATGTTTGAAAAT-3

; reverse 5

-ATTTA

AGTGGATCCAATTGGTTTC-3

), using the genomic

DNA from the Cl. acetobutylicum. The reaction oc-

curred in 50 ll of 100 mM Tris–HCl buffer (pH 8.3)

containing 50 mM KCl, 4 mM MgCl

0.4 mM of each

deoxynucleoside triphosphate, 3 lM of primer, 50 ng of

DNA, and 2.5 U of Taq polymerase (Beckman, Fuller-

ton, CA). Amplification was performed in a DNA

thermal cycler (Perkin–Elmer Cetus, Norwalk, CT)

programmed for 3 min at 92 °C and 25 cycles of 1 min at

92 °C, 1 min at 50 °C, and 1 min at 72 °C. In the end,

final cycle of 30 min at 72 °C was added. In this way, two

new restriction sites were created in the amplification

product. NdeI restriction site (in bolds in the forward

primer) was created at the 5

-end of the gene incorp-

orating the started codon ATG (underlined) at the be-

ginning of the translated sequence and a BamHI site (in

bold in the reverse primer). Amplification product was

resolved by electrophoresis in a 0.8% agarose gel

according to Sambrook [18] and was detected by stain-

ing with ethidium bromide. After agarose gel electro-

phoresis the PCR-amplified DNA fragment was purified

by selective adsorption/desorption on glass beads (Gene Clean, Bio101, La Jolla, CA, USA). The 1.5 kb DNA fragment obtained was ligated into the pGEM-T vector (Promega) named pClost1 and transformed into E. coli DH5a strain. The clones were selected and tested by restriction analysis. This plasmid was cleaved with NdeI–BamHI endonucleases and the resulting DNA fragment was ligated into plasmid PET-3a (named pClost2) cleaved with the same enzymes. This plasmid was transformed into E. coli BL-21 and cell-free extracts were obtained as above and tested for GAPDH and GAPN activities.

Purification of recombinant GAPN

The recombinant GAPN was purified to electropho- retic homogeneity from cultures of the E. coli mutant BL-21 transformed with the pClos2, using procedure involving conventional anion-exchange chromatog- raphy and hydroxyapatite chromatography. All oper- ations were performed at 4 °C.

Step 1. Cell-free extract preparation. E. coli (pClost2) strain was cultivated during 24 h in LB medium sup- plemented with Amp in presence of 1 mg ml

of IPTG to induce the expression. The cells were harvested by centrifugation at 4 °C (8000g, 10 min). Cell pellets were washed twice in 25 mM Tris–HCl buffer, pH 7.5, and resuspended in the same buffer added with 2 mM dith- iothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10% (v/v) glycerol, and 1 mM K

HPO

. The cells were then disrupted by ultrasonic treatment in a chilling water bath, using a Branson 25U sonifier at medium strength. The resulting broken-cells suspension was centrifuged at 20,000g for 20 min to obtain the crude extract.

Step 2. DEAE-cellulose chromatography. The super- natant (considered the crude extract) was applied at a flow rate of 10 ml h

to a DEAE-cellulose DE-52 (Whatman, Maidstone, England) column (3 12 cm) preequilibrated with standard buffer (25 mM Tris–HCl buffer, pH 7.5 with 10 mM 2-mercaptoethanol, 2 mM DTT, 1 mM PMSF, and 10% (v/v) glycerol). After a through wash, anion-exchange chromatography was performed with a linear gradient of potassium chloride (KCl) (0–400 mM, pH 7.5; total volume, 500 ml) in standard buffer. Fractions of 3 ml were collected and those that showed enzymatic activity were pooled and dialyzed for 24 h against standard buffer.

Step 3. Hydroxyapatite chromatography. The frac- tions were introduced at a flow rate of 1 ml h

in hy- droxyapatite column. After a through wash with standard buffer, the elution of the enzyme was per- formed with a gradient of potassium phosphate (0–200 mM; total volume of 50 ml) in standard buffer.

The fraction with maximal activity was collected and pooled.

Protein techniques

Protein concentration was estimated by the Bradford technique [19], using ovalbumin as a standard. Electro- phoresis of protein extracts was carried out according to the method described by Laemmli [20] in 12% (w/v) acrylamide slab gels in the presence of sodium dodecyl sulfate (SDS–PAGE), using Sigma MW-SDS-701 pro- tein markers using a Miniprotean-II (Bio-Rad) cuvette.

The isoelectric point of native purified GAPN was determined by analytical chromatofocusing. Column chromatofocusing in the pH range 8.0–3.0 was performed in Polybuffer Exchanger (PBE) column (1 18 cm, bed vol.) equilibrated with standard buffer. After application of the purified protein (about 0.5 mg), the column was washed with 5 ml of standard buffer. The enzyme was eluted at a flow rate of 12 ml h

by washing the column with 10 bed volumes of 10-fold-diluted Polybuffer 74-HCl adjusted to pH 3 with acetic acid, according to the speci- fication of the manufacturer (Chromatofocusing; Phar- macia fine Chemicals, Uppsala, Sweden, 1980).

The molecular mass of the native GAPN was esti- mated according to the method of Hedrick and Smith [21] using native PAGE slab gels of different acrylamide concentrations in the absence of SDS. The protein markers used were ferritine (440 kDa), catalase (232 kDa), aldolase (154 kDa), and ovalbumine (43 kDa).

The migration was made in 4 °C at 200 V. The plot LogðR

Þ versus the concentration of freezing makes it possible to give the calibration curve.

Immunological techniques

A rabbit was injected with 800 lg of GAPN from puri- fied recombinant protein in aqueous solution 1:1 with Freund’s coadjuvant. After 21 days, a sample of blood was collected, and a second dose of 500 lg of the protein was injected. After one week, 50 ml of rabbit blood was collected and serum was separated by letting it coagulate overnight at 4 °C and then centrifuging it. The obtained serum, containing monospecific anti-GAPN polyclonal antibodies, was sampled and stored at )20 °C.

Immunoblot assays (Western blot) of protein samples were carried out after electrophoresis in SDS–12%

polyacrylamide slab gels. Proteins were electroblotted onto a nitrocellulose membrane (Bio-Rad) by using a Biometra Fast-Blot and incubated with 1:800-fold di- luted antiserum in Tris-buffered saline (TBS) with 5%

(w/v) skim milk. The membrane was then washed four

times (15 min each) in TBS plus 0.05% (v/v) Tween 20

(TBSt) and incubated for 45 min with a goat anti-rabbit

immunoglobulin G antibody-peroxidase conjugate

(1:1000; Boehringer Mannheim). After four 15 min

rounds of washing with TBSt, the nitrocellulose filter

was developed under a mixture of TBS, 2 mM H

O

,

and 10 mM 4-chloro-3-naphtol in methanol. Filters were

processed and when necessary quantified with an ana- lytical imaging instrument (BioImage; Millipore).

Results and discussion

Cloning and overexpression of the Cl. acetobutylicum nonphosphorylating NADP-dependent GAPDH (GAPN) The nonphosphorylating NADP-dependent glyceral- dehyde-3-phosphate dehydrogenase was detected in soluble fraction of Cl. acetobutylicum strain. The assays of GAPN activity, carried out with NADP, show a va- lue of 0:013 U mg

. This value is lower than those of other bacterial nonphosphorylating NADP-dependent GAPDH [22]. Cl. acetobutylicum strain also exhibited a typical phosphorylating NAD-dependent GAPDH ac- tivity (1:01 U mg

). The nonphosphorylating GAPDH activity in Cl. acetobutylicum is absolutely specific for NADP. So far a nonphosphorylating NAD-dependent activity was only described in the anaerobic archaebac- terium T. tenax [8].

The coding region of the gapN gene was amplified from Cl. acetobutylicum DNA by PCR, using two syn- thetic primers containing engineered BamHI and NdeI.

These primers were selected by analyzing the genome sequence from the bacterium Cl. acetobutylicum (Gen- Bank databases: http://www.ncbi.nlm.nih.gov/PMGifs/

Genomes/micr.html).

The 1.5 kb amplified fragment was cleaved with NdeI–BamHI endonucleases, ligated into the pGEM-T vector (pClost1) and transformed into E. coli DH5a strain. The clones were selected and tested by restric- tion analysis. The fragment of plasmidic DNA from E. coli DH5a (pClost1) was obtained by partial diges- tion with BamHI and NdeI endonucleases and the re- sulting DNA (approximately 1.5 kb) fragment was ligated into plasmid PET-3a (pClost2) cleaved with the same enzymes. The insert of plasmid pClost2 was se- quenced in both strands and found to be identical to the corresponding genomic DNA sequence. This plas- mid was transformed into E. coli BL-21 (Fig. 1) and cell-free extracts were obtained as above and tested for GAPDH and GAPN activities.

Fig. 1. Subcloning of the Cl. acetobutylicum GapN gene from the genomic DNA into the expression vector PET-3 amp, ampicilline resistant gene; ori,

origin of replication.

As shown in Table 1, the NAD-dependent activity of the typical glycolytic enzyme was found in crude ex- tracts of E. coli BL-21 strain and BL-21 harboring plas- mid (pClost2) without IPTG induction. No significant GAPN activity was observed in these strains. In con- trast, crude extracts from the E. coli BL-21 harboring plasmid (pClost2) with induction also exhibited higher level of the nonphosphorylating NADP-dependent ac- tivity (GAPN). The fact that cell extract of the recom- binant clone exhibited a specific activity level 170-fold higher than those found in natural Cl. acetobutylicum strain indicates that the GAPN was indeed overex- pressed in the recombinant clone.

A high transcription level of the GapN gene and consequently a high production of the 50-kDa GAPN protein were expected in E. coli cells in the presence of the IPTG inducer. As shown in the SDS–polyacryl- amide gel electrophoresis (SDS–PAGE) of total protein

preparation, a predominant protein band 50-kDa was observed in extracts from E. coli BL-21 (pClost2) after 24 h IPTG induction. A minor 50-kDa band was de- tected in total protein of the natural strain, whereas no corresponding band was observed in the non-trans- formed E. coli BL-21 and in uninduced BL-21 (pClost2) strain (Fig. 2A). As judged by densitometry of the SDS–PAGE gels, the GAPN accounted for about 40% of the total protein present in the soluble protein fraction after sonication of the induced BL-21 (pClost2) cells.

No noticeable 50-kDa protein band was found on SDS–PAGE or immunoblot analyses of the pellet frac- tion of the E. coli BL-21 cells harboring plasmid pClost2 (data not shown), suggesting that most of the heterol- ogous expressed GAPN was recovered in the soluble protein fraction.

Purification of the recombinant GAPN

The purification of the recombinant NADP non- phosphorylating GAPDH was first achieved with a classical protocol using ammonium sulfate precipitation, chromatography on DEAE cellulose, hydrophobic chromatography on phenyl-Sepharose and blue-Sepha- rose [9]. We used a simplified purification protocol without ammonium sulfate precipitation and hydro- phobic chromatography steps because we noted that ammonium sulfate inhibits irreversibly Cl. acetobutyli- cum GAPN activity.

The IPTG-induced NADP nonphosphorylating GAPDH has been purified about 34-fold from E. coli

Table 1

Enzymatic activity (U mg

) of the non-phosphorylating NADP-de- pendent GAPDH (GAPN) and the phosphorylating NAD-dependent GAPDH (GAPc) in the crude extract of Cl. acetobutylicum and E. coli

Enzymatic activity of the GAPN ðU mg

Þ

Enzymatic activity of the GAPc ðU mg

Þ

Cl. acetobutylicum 0.013 1.01

E. coli BL-21 <0.001

0.098

E. coli BL-21 (pClost2) without IPTG-induction

<0.001

0.096

E. coli BL-21 (pClost2) after 24 h of IPTG-induction

2.20 0.095

a

Not detected.

Fig. 2. (A) Coomassie blue-stained SDS–PAGE electrophotogram showing the protein patterns of cell-free protein extracts. Superexpression of

GAPN from Cl. acetobutylicum in E. coli BL-21; lane a, Cl. acetobutylicum strain; lane b, BL-21 no transformed; lane c, BL-21 transformed with the

plasmid pClost2 without IPTG-induction; lane d, BL-21 transformed with the plasmid pClost2 after 24 h of IPTG-induction. About 60lg of protein

was loaded per lane in all cases. The positions and molecular masses of marker proteins are indicated. (B) Immunoblot analysis of cell-free protein

extracts from organisms using monospecific antibodies. lane a, Cl. acetobutylicum strain; lane b, BL-21 no transformed; lane c, BL-21 transformed

with the plasmid pClost2 without IPTG-induction; lane d, BL-21 transformed with the plasmid pClost2 after 24 h of IPTG-induction. About 60lg of

protein was located per lane.

BL-21 cells harboring plasmid (pClost2), using a straightforward procedure involving ion-exchange and hydroxyapatite chromatographies.

Table 2 summarizes a representative purification.

Values of 75 U mg

protein were obtained for the puri- fied enzyme, with yield of 43%. Ion-exchange chromatog- raphy step represented by far the largest contribution to the overall purification (purification factor was 29.2), because during this step most of other proteins were re- moved from the enzyme preparation. At this stage, the enzyme preparation still contained, as checked by SDS–

PAGE, a few contaminating proteins (Fig. 3, lane b) which were eventually separated from GAPN by hy- droxyapatite chromatography. The enzyme activity was eluted from hydroxyapatite column as a symmetrical peak. This procedure was the first report of hydroxyapa- tite column used for GAPN purification. It was suc- cessfully used for the purification of the recombinant Cl.

acetobutylicum GAPN.

SDS–PAGE analysis of the different fractions ob- tained during the purification procedure showed a pro- gressive enrichment of a 50-kDa protein (Fig. 3). Only this protein band, which corresponds to the non- phosphorylating NADP-dependent subunit, was ob- served in the electrophoretically homogeneous final enzyme preparation (Fig. 3, lane c). The very minor protein band observed on the SDS–PAGE of the puri- fied GAPN should be an artefact, since it is also seen on the SDS–PAGE of the single enzyme peak obtained after chromatofocusing of the purified preparation (see photograph in Fig. 4). Moreover, no other significant protein peak was found by this chromatographic pro- cess (Fig. 4).

A polyclonal antibody raised against purified re- combinant Cl. acetobutylicum GAPN was used for Western blot analysis. This antibody clearly recognized a single protein band of 50-kDa, corresponding to the GAPN subunit, in protein extracts either from Cl.

acetobutylicum or from E. coli BL-21 bearing pClost2 (Fig. 2B). No band was observed in crude extract of wild type E. coli K-12.

Physicochemical studies of recombinant GAPN

Concerning the physicochemical parameters of the purified protein, Hedrick method [21] using non-dena- turing acrylamid gels, yielded values of 204 kDa for the native molecular mass (data not shown). SDS–PAGE of

the recombinant GAPN showed, as stated above, a single stained band corresponding to a 50-kDa protein (Fig. 3, lane c), thus indicating that Cl. acetobutylicum non- phosphorylating NADP-dependent GAPDH should have a homotetrameric structure. Since active enzyme is homotetramer determined from beet, Ch. reinhardtii and H. brasiliensis [12–14] and St. mutans [23].

Column chromatofocusing, a chromatographic technique of protein separation according to pI’s, showed a single protein peak which perfectly over- lapped with that of GAPN activity (Fig. 4). Maximal values of both parameters (absorbance and activity) were found at pH 4.2, which corresponding to the pI of the enzyme estimated by this method. The acid pI value of the Cl. acetobutylicum GAPN is different from basic pI of GAPN in higher plants [12,13] in- dicating that acid pI is probably typical for the bac- terial GAPN. Since chromatofocusing has been proved to efficiently resolve closely related GAPDH isoforms in other organisms [24,25], this result indicates the presence of only one GAPN isoform in Cl. acetobu- tylicum. A single GAPN isoform has been found also in other organisms, both bacteria and eukaryotes [7,12,13].

The optimal pH value for the oxidative reaction was 8.2 with considerable activity in the range of 5–13.

Preincubation of Cl. acetobutylicum GAPN for 10 min at temperatures varying 15–65 °C range did not affect irreversibly the enzyme activity. Thermal inactivation did, however, occur above 70 °C and resulted in total activity loss at 95 °C. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 65 °C with activation energy 18 KJ mol

calculated using the corresponding linear Arrhenius plot (data not shown).

Catalytic properties of recombinant GAPN

The Cl. acetobutylicum GAPN uses both

and

L

L

-form of G3P in the reaction. In contrast, the GAPDH:NADP oxidoreductase of plant and green alga was absolutely specific for

-form of G3P [26] indicating a possible difference in the enzyme structures of these two enzymes. From double-reciprocal plots of reaction rates under standard assay conditions in the presence of saturating concentrations of one substrate, apparent K

-values for NADP and

-G3P or

-G3P were estimated to be 0:200 0:05 and 0:545 0:1 mM,

Table 2

Purification of recombinant Cl. acetobutylicum GAPN from E. coli BL-21 transformed with pClost2

Fraction Total protein (mg) Total activity (U) Specific activity (U mg

) Factor (fold) Yield (%)

Crude extract 1650 3630 2.2 1 100

DEAE-cellulose eluate 29.4 1888 64.2 29.2 52

Hydroxyapatite eluate 20.8 1561 75 34 43

respectively. This result was completely different with K

obtained for St. mutans GAPN [27]. The V

of the purified protein was estimated to be 78:8 U mg

.

The Cl. acetobutylicum GAPN was markedly inhib- ited by 1 mM sulfhydryl-modifying reagent iodoacet- amide (76%), thus showing that this inactivation occurs through oxidation of vicinal dithiols [28]. This inhibition was prevented by both NADP and

-G3P. These results suggest the participation of essential sulfhydryl groups in the catalytic activity of G3P:NADP oxidoreductase.

These results are the first to be reported on cloning, purification, and characterization of non-phosphorylat- ing NADP-dependent GAPDH from Cl. acetobutylicum strain. The procedure used for the GAPN purification was rapid and straightforward involving two chroma- tographic steps, namely ion-exchange and hydroxyapa- tite chromatographies.

Acknowledgments

This work is part of a Collaborative Research Project between CNR (Morocco) and CSIC (Spain). This work was supported by AECI (Spain) a collaborative grant from the Andalusian Government — Minist eere d’Educa- tion et de la Recherche Scientifique’ of Morocco (Junta de Andaluc ııa, Convenio Colaboraci o on Univ. Marroqu ııes), group PAI CVI-261 and PARS (Morocco). Authors thank Prof. Manuel Losada for his generous encourage- ment and MCYT of Spain (Grants PB 97-1135 and BCM 2001-563) for additional support. We thank the two re- viewers for their many helpful suggestions.

References

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[3] H. Brinkman, R. Cerff, M. Salomon, J. Soll, Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3- phosphate dehydrogenase from pea and spinash, Plant Mol. Biol.

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[5] A. Serrano, M.I. Mateos, M. Losada, ATP-driven transhydrogen- ation and ionization of water in a reconstituted glyceraldehyde–

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[6] A. Brown, L. Wittenberger, The occurrence of multiple glyceral- dehyde-3-phosphate dehydrogenases in caryogenic streptococci, B. B. R. Commun. 43 (1971) 217–224.

[7] D.A. Boyd, D.G. Cvitkovitch, I.R. Hamilton, Sequence, expres- sion, and function of the gene for the non-phosphorylating, Fig. 3. Coomassie Blue-stained SDS–PAGE electrophotogram show-

ing different purification step fraction. Lane a, cell-free protein ex- tracts; lane b, anion-exchange chromatography eluat pool; lane d, hydroxyapatite chromatography eluate pool (pure protein prepara- tion). Purification of GAPN from BL-21 strain transformed with pClost2. About 60lg of protein was located in lane a; 40 lg in lanes b and c. The arrow points to the 50-kDa GAPN submit band.

Fig. 4. Analytical chromatofocusing on Polybuffer Exchanger 74 of purified GAPN from E. coli BL-21 strain holding the GapN of Cl.

acetobutylicum. A sample containing about 0.5 mg of native protein

was applied to Polybuffer Exchanger 74 column (1 18 cm bed vol.)

equilibrated with 25 mM Tris–HCl buffer, pH 8.0 containing 0.5 mM

EDTA, and 10 mM-2-mercaptoethanol. The enzyme was eluted by

using a pH gradient of Polybuffer 74/HCl, pH 3.0. Fractions (1.5 ml)

were collected. The photograph shows the Coomassie blue-stained

SDS–PAGE electrophotogram of the activity peak fractions after

chromatofocusing. The arrow indicates the 50-kDa protein band cor-

responding to the GAPN subunit. About 40 lg of protein was located

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