Cloning, gene expression and characterization of a novel bacterial NAD-dependent non-phosphorylating glyceraldehyde-3-
phosphate dehydrogenase from Neisseria meningitidis strain Z2491
Latifa FourratÆAbdelghani IddarÆFederico Valverde Æ Aurelio SerranoÆ Abdelaziz Soukri
Received: 28 March 2007 / Accepted: 21 June 2007 / Published online: 10 July 2007 Springer Science+Business Media B.V. 2007
Abstract Alignment of the amino acid sequence of some archaeal, bacterial and eukaryotic non-phosphorylating glyceraldehydes-3-phosphate dehydrogenases (GAPNs) and aldehyde dehydrogenases (ALDHs) with the sequence of a putative GAPN present in the genome of the Gram- negative bacterium Neisseria meningitidis strain Z2491 demonstrated the conservation of residues involved in the catalytic activity. The predicted coding sequence of theN.
meningitidis gapN gene was cloned in Escherichia coli XL1-blue under the expression of an inducible promoter.
The IPTG-induced GAPN was purified ca. 48-fold from E. colicells using a procedure that sequentially employed conventional ammonium sulfate fractionation as well as anion-exchange and affinity chromatography. The purified recombinant enzyme was thoroughly characterized. The protein is a homotetramer with a 50-kDa subunit, exhibit- ing absolute specificity for NAD and a broad spectrum of aldehyde substrates. Isoelectric focusing analysis with the purified fraction showed the presence of an acidic poly- peptide with an isoelectric point of 6.3. The optimum pH of
the purified enzyme was between 9 and 10. Studies on the effect of increasing temperatures on the enzyme activity revealed an optimal value ca. 64C. Molecular phyloge- netic data suggest thatN. meningitidisGAPN has a closer relationship with archaeal GAPNs and glyceraldehyde de- hydrogenases than with the typical NADP-specific GAPNs from Gram-positive bacteria and photosynthetic eukary- otes.
Keywords Neisseria meningitidisNon-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) NAD(P)-dependencyAldehyde dehydrogenase superfamily
Introduction
The Gram-negative bacteriumNeisseria meningitidisis an obligate human pathogen that inhabits the upper respiratory tract, from which it occasionally disseminates, causing disease. Invasion results in bacteraemia, with possible progression to sepsis, meningitis, and death. Cerebrospinal meningitis remains a devastating disease worldwide, with a morbidity of 1–3 per 100,000 in North America and Europe and considerably higher in poorer regions, such as Africa [1]. The mechanisms by which the pathogenN. meningit- idis passes from the site of initial colonization, the naso- pharyngeal mucosa, to invade the cerebrospinal fluid are not well understood. In fact, carbon metabolism strongly influences the pathogenic capacity of this fastidious bac- terium [2]. Therefore, the description of new enzymes in- volved in carbon metabolism of N. meningitidismay have important biomedical implications.
Glycolysis is the main pathway for carbohydrate deg- radation in nearly all organisms. The generation of the final L. FourratA. Soukri (&)
Laboratoire de Physiologie et Ge´ne´tique mole´culaire, De´partement de Biologie, Faculte´ des Sciences Aı¨n-Chock, Universite´ Hassan-II, Casablanca, Morocco
e-mail: [email protected] A. Iddar
Unite´ Radio-Immuno-Analyse, De´partement des Sciences du vivant, Centre National de l’Energie, des Sciences et des Techniques Nucle´aires (CNESTEN), Centre d’Etudes Nucle´aires de la Maaˆmora, Rabat, Morocco
F. ValverdeA. Serrano
Instituto de Bioquı´mica Vegetal y Fotosı´ntesis
(CSIC-Universidad de Sevilla), Centro de Investigaciones Cientı´ficas Isla de la Cartuja, Sevilla, Spain
DOI 10.1007/s11010-007-9545-z
product of glycolysis from glucose, pyruvate, is completed by nine enzymatic steps, most of which function in the reverse direction during gluconeogenesis. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme but can also be involved in other central pathways of carbon metabolism [3]. The most common member of the GAPDH family is the NAD-dependent enzyme (EC 1.2.1.12) found in all cellular organisms so far studied and located in the cytosol. This enzyme plays a crucial role in the Embden–Meyerhoff pathway, not only in glycolysis but also in gluconeogenesis [3]. The NADP-dependent GAP- DH isoform (EC 1.2.1.13), located in the chloroplast stroma and the cyanobacterial cytosol, is involved in photosynthetic CO2 assimilation [4–6]. Both enzymes catalyze the same reversible reaction, the oxidation ofD- glyceraldehyde-3-phosphate (G3P) to 1,3-D-bisphospho- glycerate (1,3-BPGA) with the concomitant addition of a molecule of Pi, but they are encoded by distinct, although related, nuclear genes (GapC and GapA/B, respectively), differ in the coenzyme specificity and are located in dif- ferent cellular compartments [5, 7]. The non-phosphory- lating glyceraldehyde-3-phosphate dehydrogenase (GAPN;
EC 1.2.1.9) is encoded by the nucleargapN gene in pho- tosynthetic eukaryotes, from which it was first cloned, thus allowing the identification of this protein as a member of the aldehyde dehydrogenase superfamily [8]. GAPN cata- lyzes the oxidation of G3P to 3-phosphoglycerate (3-PGA) with the reduction of NADP to NADPH. No inorganic phosphate is required and the reaction is irreversible under physiological conditions. It was originally reported to be exclusively present in green microalgae and plants, where the enzyme was proposed to metabolize trioses exported from the chloroplast, although its precise function remains yet to be established [9, 10]. This was followed by the discovery of GAPN activity in other photosynthetic eukaryotes, e.g., in different algae [9, 11]. However, a NADP-dependent non-phosphorylating GAPDH was cloned, purified and characterized first fromStreptococcus mutans [12] and later from Streptococcus pyogenes and Clostridium acetobutylicum [13, 14]. NADP-dependent GAPN was recently reported to be widespread among Gram-positive bacteria of different phylogenetic groups (Streptococcaceae, Clostridia, and Bacillaceae) [15]. A systematic survey of GAPN activities among different groups of bacteria showed that N. meningitidis, lacked a non-phosphorylating NADP-dependent GAPDH activity [15]. By contrast, the archaeonPyrococcus furiosus pos- sesses a GAPOR activity (ferredoxin-dependent GAP oxi- do-reductase), but the sequence of the enzyme is distinctly separated from that of NADP-dependent GAPNs [16].
Other GAPN class was described in hyperthermophilic archaea Thermoproteus tenax [17] and Sulfolobus solfa- taricus[18].It was identified as an integral constituent of a
catabolic Embden–Meyerhof–Parnas (EMP) pathway in these organisms. Sequence analysis of GAPN fromT. tenax revealed that the enzyme belongs to the aldehyde dehy- drogenase superfamily (ALDH; E.C. 1.2.1.3) and shows the highest sequence similarities to the non-phosphorylat- ing GAPDHs [17]. Interestingly, the enzymatic properties of the T. tenax GAPN suggest a significantly different physiological context, adding new aspects to the structural and functional plasticity within the ALDH superfamily [8].
The purified protein is a homotetramer of 204 kDa exhib- iting specificity for NAD (as previously assumed) [17] but Early studies indicated that NADP was the preferred co- substrate in presence of activators (e.g., glucose-1P) and only in the absence of activators NAD was used [19].
The complete genome sequence of N. meningitidis serotype A strain Z2491 has been determined [20, http://
www.ncbi.nlm.nih.gov/genomes/static/eub.html]. From this sequence, we cloned the ORF of a potentialgapNgene (annotated as an aldehyde dehydrogenase A gene) encod- ing a putative non-phosphorylating GAPDH. In this work, we report the cloning of theN. meningitidis gapNgene as well as the expression, purification and characterization of the encoded protein, which is the first bacterial NAD- dependent GAPN reported so far. The phylogenetic and physiological implications of this discovery are discussed herein.
Materials and methods
Chemical and plasmids
D-G3P was prepared from monobarium salts of the diethyl acetal (Sigma); all other chemicals (analytical grade) were from Fluka or Merck. Cloning of PCR products and protein expression were performed using the plasmids pGEM-T (Promega) and pTrc 99A (Pharmacia Biotech), respec- tively.
Organisms and growth conditions
Neisseria meningitidis strain Z2491 (serogroup A, ST-4), whose complete genome has been sequenced [20], was used for DNA isolation or for preparation of cell extracts.
This strain was grown on GCB medium (Difco Laborato- ries, USA) [21].
Escherichia colistrain XL1-blue and transformed XL1- blue clones were grown at 37C in Luria-Broth (LB) [22].
Either solid (plus 1.5%, w/v, Difco-Bactoagar) or liquid cultures were used. When necessary, ampicillin (Amp) and isopropyl-b-thiogalactopyranoside (IPTG) were added at concentrations of 100lg ml–1 and 1 mg ml–1, respec- tively.
Enzyme assays
The non-phosphorylating GAPDH activity was measured spectrophotometrically as described in [23]. The reaction was started by adding the enzyme to the assay mixture containing 50 mM Tricine buffer (pH 8.5), 2 mM NAD or NADP, and 2 mM D-glyceraldehyde-3-phosphate (D-G3P) at 25C. Absorbance changes at 340 nm were followed.
The phosphorylating NAD-dependent GAPDH activity was measured using the same procedure with 10 mM sodium orthophosphate (PO43–) or arsenate (AsO43–) in the reaction medium. Dehydrogenase activity with other aldehydes was also determined spectrophotometrically at 340 nm. One enzymatic activity unit was defined as the amount of en- zyme that reduces 1lmol NAD(P) min–1 at 25C. The reaction mixture contained 50 mM Tricine buffer (pH 8.5), 0.1 M KCl, 1.5 mM NAD or NADP, 1.2 mMD,L-glycer- aldehyde or acetaldehyde, and enzyme. Kinetic constants were calculated from initial rates. For the determination of kinetic parameters, the concentrations of the respective fixed substrate for the reaction were 1 mM NAD or 3 mM
D-G3P. Km andVmax were determined from Lineweaver–
Burk double-reciprocal plots. The optimal pH was esti- mated in the pH range from 5 to 13 using different buffers (acetate, imidazole, Tris and carbonate/bicarbonate) ad- justed to the same ionic strength as the standard mixture.
Temperature effect was determined by measuring the activity in 50 mM Tricine buffer within a temperature range from 10 to 90C after adjusting pH to 8.5 at different temperatures. Influence of possible effectors onNeisseria GAPN was investigated by measuringKmandVmaxchan- ges with increasing concentrations of effectors. The metabolites tested were NADPH, NADH and glucose-1- phosphate (in the range 0.05–1 mM); AMP, ADP, ATP and fructose-6-phosphate (in the range 2–20 mM).
DNA manipulation and cloning
Chromosomal DNA was isolated from N. meningitidis strain Z2491 using the WIZARD Kit (Promega). A PCR amplification of a 1.45-kb DNA fragment, corresponding to a ORF annotated as an aldehyde dehydrogenase A gene (CAB83774) in the strain Z2491 genome, was carried out at 50C employing two strict oligonucleotides (forward, 5¢- GGA GAA CCT GCC ATG GAA CAA TTG GCC-3¢;
reverse, 5¢-CGC GGA TCCTTA AAT GTC GGT TTC C-3¢, using the genomic DNA from theN. meningitidisas a template.The 50ll reaction was carried out in 100 mM Tris–
HCl buffer (pH 8.3) containing 50 mM KCl, 4 mM MgCl2, 2ll of dimethyl sulfoxide (DMSO), 0.4 mM dNTPs, 3lM of each primer, 50 ng of genomic DNA and 2.5 U of GoTaqR DNA Polymerase (Promega). Amplification was performed in a DNA thermal cycler (Primus, MWG-BIOTECH)
programmed for 2 min at 92C and 35 cycles of 1 min at 92C, 1 min at 50C and 1 min at 72C. A final cycle of 30 min at 72C was added. In this way, two new restriction sites were created in the amplification product: a newNcoI restriction site (in bold in the forward primer) at the 5¢-end of the gene incorporating the start codon ATG (underlined) at the beginning of the translated sequence, and aBamHI site (in bold in the reverse primer) after the stop codon (underlined). The 1.4 kb DNA fragment thus obtained was ligated into the pGEM-T vector (Promega), named pNeis1 and transformed intoE. coliDH5astrain. Vectors rescued from some selected clones were tested by restriction analysis. Plasmids were further restricted withNcoI-BamH I endonucleases and the resulting 1.4 kb DNA fragment ligated into plasmid pTrc 99A previously restricted with the same enzymes. The final construct (named pNeis2) was sequenced in both strands and found to be identical to the corresponding genomic DNA sequence. This plasmid was then transformed into E. coli XL1-blue and cell-free ex- tracts were obtained as above and tested for GAPDH and GAPN activities.
Purification of recombinant GAPN
The recombinant N. meningitidis GAPN was purified to electrophoretic homogeneity from cultures of the E. coli mutant XL1-blue transformed with the pNeis2, by a pro- cedure modified from that described for theT. tenaxGAPN [17].
Step 1: Cell-free extract preparation
Escherichia coli (pNeis2) strain was cultured at 37C and 200 rpm shaking in 2 l of LB medium with Ampicilin (100 lg ml–1), to an OD of 0.6 at 600 nm. IPTG (1 mg ml–1) was added and the culture was grown for 4 h more at 37C to inducegapNexpression. Cells were harvested by centrifu- gation at 4C (8,000·g, 10 min), cell pellets were washed twice in 25 mM Tris–HCl buffer, pH 7.5, and resuspended in the same buffer supplemented with 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10% (v/v) glycerol.
The cells were then disrupted by sonication in a chilling water bath, using a Branson model B12 Sonifier at medium strength. The resulting broken-cell suspension was centri- fuged at 20,000· gfor 20 min at 4C to obtain the crude extract.
Step 2: Ammonium sulfate precipitation
The supernatant (considered the crude extract) was treated with solid ammonium sulfate at 4C. The protein fraction resulting from a fractionation with 35–85% (w/v)
ammonium sulfate saturation was collected. This protein pellet was resuspended in a minimal volume of standard buffer (25 mM Tris–HCl buffer pH 7.5 with 2 mM EDTA, 1 mM PMSF and 10% (v/v) glycerol) and dialyzed over- night against 5 l of the same buffer at 4C.
Step 3: DEAE-cellulose chromatography
The extract was applied at a flow rate of 30 ml h–1 to a DEAE-cellulose DE-52 (Whatman, Maidstone, England) column (2 by 10 cm) preequilibrated with standard buffer.
Elution was performed with a linear gradient of potassium chloride (KCl) (0–300 mM pH 7.5; total volume of 200 ml) in standard buffer. Fractions of 2 ml were col- lected and those with enzymatic activity were pooled.
Step 4: Dye-affinity chromatography on Cibacron Blue Sepharose
The pooled fractions were dialyzed against standard buffer and introduced at a flow rate of 10 ml h–1in Cibacron Blue Sepharose CL-6B column (1 by 5 cm) equilibrated with two bed volumes of the same buffer. The column was washed with five volumes of standard buffer and the en- zyme collected with a pulse (1–2 ml) of buffer containing 5 mM NAD at a flow rate of 10 ml h–1. To remove NAD from the enzyme preparation, the active pool was further dialyzed against standard buffer.
Protein techniques
Protein concentration was estimated by the Bradford technique [24], using ovalbumin as a standard. Electro- phoresis of protein extracts was carried out according to [25] in 12% (w/v) acrylamide slab gels in the presence of sodium dodecyl sulfate (SDS-PAGE), using a pre-stained SDS-PAGE molecular mass protein standards (Precision Plus ProteinTM; Bio-Rad) and a Miniprotean-II (Bio-Rad) cassette.
Isoelectric focusing was performed with the same electrophoretic system above described in 5% (w/v) poly- acrylamide slab gels holding ampholyte-generated pH gradients (pH range 3.5–10.0; carrier ampholyte for IEF pH 3.5–10; SIGMA). 25 mM NaOH and 20 mM CH3COOH were used as cathode and anode solutions, respectively.
The pI protein markers kit used was the Bio-Rad IEF standards pIrange 4.45–9.6 for isoelectric focusing.
The molecular mass of the native GAPN was estimated by running native PAGE slab gels with varying acrylamide concentrations (5–10% (w/v)) in the absence of SDS [26].
The protein markers used were ferritine (440 kDa), cata- lase (232 kDa), aldolase (154 kDa), and ovalbumin
(43 kDa). Electrophoresis was conducted at 4C and 200 V. A plot of log of relative mobility (Rf) versus acrylamide concentration allowed us to obtain a linear calibration of molecular masses.
Immunochemical techniques
A rabbit was injected with 800 lg of purified recombinant GAPN protein in aqueous solution 1:1 together with Fre- und’s coadjuvant. After 21 days, a sample of blood was collected, and a second dose of 500lg protein was in- jected. After 1 week, 20 ml of rabbit blood was collected and serum was separated by letting it coagulate overnight at 4C and then centrifuging it. The serum, containing monospecific anti-GAPN polyclonal antibodies, was sam- pled and stored at –20C.
Immunoblot assays (Western blot) of protein samples were carried out after electrophoresis in 12% (w/v) poly- acrylamide SDS-PAGE slab gels. Proteins were electrob- lotted onto a nitrocellulose membrane (Bio-Rad) with a Biometra Fast-Blot system and incubated with 1:800-fold diluted antiserum in Tris-buffered saline solution (TBS) plus 5% (w/v) skimmed 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 conju- gate (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 H2O2, and 10 mM 4-chloro-3-naphtol in methanol. Filters were pro- cessed and when necessary quantified with an analytical imaging instrument (BioImage; Millipore).
Database searches, sequence and phylogenetic analyses BLAST searches were made employing the National Center for Biotechnology Information Website facilities (http://www.ncbi.nlm.nih.gov/). Multiple sequence align- ments of GAPN partial sequences were done with the Clustal X v.1.8 program [27]. These sequences correspond to an internal region from the putativegapNgene fromN.
meningitidis strain Z2491 and other selected protein se- quences available in databases and includes the conserved active site Cys-containing motif. Phylogenetic trees were constructed from this protein alignment with the distance (neighbor-joining, Kimura distance calculations) and max- imum likelihood methods using the programs Clustal X v.1.8 and Tree-Puzzle v.5.0 [28], respectively, and then visualized using TREEVIEW v.1.6.6 (R.D.M. Page, 2001; http://
taxonomy.zoology.gla.ac.uk/rod/rod.html). Published amino acid sequences used in this work belonged to anno- tated putative (Methanothermobacter thermautotrophicus,
GenBank accession no. AAB85474; Methanocaldo- coccus jannaschii, Q58806; Pyrococcus furiosus, NP_578484; Thermococcus kodakaraensis, BAD84898;
Halobacterium sp., AAG19367) and experimentally vali- dated (Thermoproteus tenax, CAA71651) archaeal GAPNs.
It also included archetypical NADP-dependent GAPN from Gram-positive bacteria and photosynthetic eukaryotes (Clostridium acetobutylicum ATCC824, AJ880320; Clos- tridium difficile, AJ880325; Clostridium prefringens, AJ880321; Clostridium pasteurianum, AJ880322; Strepto- coccus pyogenes, AJ880326; Streptococcus mutans, NP_721104; Streptococcus pneumoniae TIGR4, NP_345590;Streptococcus sp., AJ880316,Bacillus thurin- giensis, AJ880324; Bacillus cereus, AJ880318; Bacillus anthracis, AAP24851;Bacillus halodurans, E83929;Pisum sativum, P81406; Nicotina plumbaginifolia, P93338; Zea mays, Q43272; Scenedesmus vacuolatus, CAC81014;
Ostreococcus tauri, CR954206.02;Cyanidioschyzon mero- lae, CMT034C). Other sequences used were: the putative gapNortholog of N. meningitidis MC58, AAF42297; my- coplasm annotated putative gapN sequences (Ureaplasma urealyticum, AAF30771; Mycoplasma capricolum, CAA83756); two archaeal glyceraldehyde dehydrogenases fromThermoplasma acidophilum (CAC11938) and Picro- philus torridus (AAT42917); and a number of ALDHs sequences from archaea, bacteria and eukaryotes (Methan- osarcina barkeri ALDH Mbar_A2387, AAZ71308; Met- hanosarcina barkeri ALDH Mbar_A0503, AAZ69485;
E. coli ALDH AAA23428; Saccharomyces cerevisiae ALDH, CAA78962; Saccharomyces cerevisiae mitochon- drial ALDH precursor, AAA34419; plant (Arabidopsis thaliana) ALDH1a, AAM27004; mammalian ALDH1, NP_071852).
Results and discussion
Comparison of the protein sequence encoded by a putativegapNgene ofN. meningitidisstrain Z2491 with homologous sequences from bacteria, archaea and eukaryotes
Bioinformatic searches identified a putative gapN gene, encoding an acidic protein (480 aa, 52.3 kDa) with strong similarity to GAPN sequences, which was annotated as an aldehyde dehydrogenase Agene (CAB83774) in the gen- ome ofN. meningitidisserotype A strain Z2491 [20,http://
www.ncbi.nlm.nih.gov/genomes/static/eub.html]. Multiple sequence alignment of an internal region (ca. 240 aa) containing conserved motifs of putative and validated GAPNs from various eukaryotic and prokaryotic sources and ALDHs showed that a number of essential residues
involved in catalysis, as well as motifs involved in sub- strate binding, are conserved in the N. meningitidis de- duced sequence (Fig.1). Noteworthy, it showed a high similarity with archaeal GAPNs, among which the Ther- moproteus tenaxGAPN has been characterized. Thus, the important residues for the active site, Glu-263 and Cys-297 (numbering according toT. tenaxsequence [17], involved in deacylation through activation and orientation of the attacking water molecule in GAPN [29–31] and in catalytic thioester formation, respectively, are strictly conserved in all sequences. This is in agreement with previous reports that describe them as essential for ALDH and GAPN activities [8,15,17]. In addition, all residues involved in NAD/NADP binding are conserved in theN. meningitidis sequence: Asn-162, Thr-239, Gly-240, Leu-264, Gly-265, and Phe-397 (see Fig. 1). The structural relationship of the N. meningitidisprotein with ALDHs and GAPNs is further confirmed by comparing the computer-modeled three- dimensional structure of this GAPN and the resolved structure of rat liver ALDH3 [32]. Thus, the basic struc- tural features of the members of the GAPN family have been conserved throughout their evolution. Nevertheless, Arg-301, necessary for the correct positioning of D-G3P into the active site of GAPNs [30,31,33] is not conserved in Neisseria enzyme and this could explain the relatively highKm for D-G3P observed for this GAPN as compared with other aldehyde substrates (see below). The highly conserved Gly-248 was also present in the ALDHs, showing that GAPNs and ALDHs probably shared a common ancestor [8]. ALDHs and GAPNs showed no significant identity match with phosphorylating GAPDHs [34]. It is interesting to note that the highest overall identity (ca. 40%) was observed between the N. meningitidis GAPN and T. tenax enzyme, characterized as non-phos- phorylating NAD/NADP-dependent GAPN. Overall, these data show thatN. meningitidisGAPN should be a member of a novel bacterial group belonging to the ALDH super- family.
Expression of the putativeN. meningitidis gapN gene encoding a NAD-dependent GAPN
The GAPN of the archaeon T. tenax had been previously cloned and successfully expressed in E. coli [17]. In this paper, a similar strategy, involving PCR amplification and eventual cloning in an expression plasmid under a strong promoter has been used for functional and structural studies on the putative GAPN fromN. meningitidis strain Z2941 as described in the Material and Methods section.
The E. coli XL1-blue strain transformed with the re- combinant expression vector pNeis2 and induced with 1 mM IPTG accumulated a new prominent soluble protein
exhibiting in SDS-PAGE and Western blot analyses an apparent molecular mass ofca. 50 kDa (data not shown).
Table1shows that the typical phosphorylating GAPDH activity due to glycolytic GAPDH enzyme was found in
crude extracts of non transformed E. coliXL1-blue cells and uninduced cells harboring plasmid pNeis2. In contrast, no significant GAPN activity was observed in these strains.
Crude extracts from the inducedE. coliXL1-blue harbor- ing plasmid pNeis2 also exhibited high levels of non- phosphorylating NAD-dependent activity (0.5–0.6 U mg–1 protein). The cell-free extract of the recombinant clone exhibited specific activity levels 65-fold higher than those found in the wild type N. meningitidis strain. The result indicates that the GAPN was indeed overexpressed in the recombinant bacterial clone.
Purification and characterization of recombinant N. meningitidisGAPN
Since most of the recombinant enzyme was present in the soluble fraction, we proceeded to its purification from the transformed E. coli clone selected as described under Fig. 1 Multiple sequences alignment using the CLUSTAL X
program of a partial protein sequence (internal region ofca. 240 residues containing conserved active site motifs) of the putative N. meningitidisGAPN and various ALDHs and GAPNs from archaea, bacteria and photosynthetic eukaryotes, as described in Materials and
methods. Important residues for catalysis and substrate binding are in bold and active site motifs are underlined. (*) indicate strictly conserved residues, among them the essential active-site located Cys- 302. (Source: UniProtKB/Swiss-Prot entries P81406, P93338, O57693, Q59931 and P32872, and references [41] and [43])
Table 1 Specific activity levels (U mg–1) of NAD-dependent GAPN and NAD-dependent phosphorylating GAPDH in crude extracts ofN.
meningitidisstrain Z2491 and transformedE. coliclones GAPN specific
activity (U mg–1)
GAPDH specific activity (U mg–1) Neisseria meningitidis
strain Z2491
0.008 2.160
E. coliXL1-blue NDa 1.010
UninducedE. coli XL1-blue (pNeis2)
NDa 1.050
InducedE. coliXL1- blue (pNeis2)
0.520 1.000
a ND, not detected (<0.001 U mg–1)
Materials and Methods. Briefly, 2 l of LB culture of the E. coli clone transformed with plasmid pNeis2 were induced with IPTG. The cell lysate obtained by sonication was subjected to centrifugation (20,000·gfor 20 min) to obtain a cell-free extract (soluble protein fraction) that was used as the starting material for enzyme purification. The supernatant was treated with solid ammonium sulfate from 35% to 85% (w/v), and the protein pellet was resuspended in 5 ml of standard buffer and dialyzed. This preparation was loaded onto a DEAE-52 anion exchange column and the enzyme was eluted with an increasing linear gradient of sodium chloride (0–0.3 M). NAD-dependent GAPN activity eluted as a single peak. SDS–PAGE analysis (Fig.2A, lane c) revealed a prominent polypeptide ofca.
50 kDa in the main fraction of the activity peak. Active fractions were dialyzed and applied to a Cibacron Blue Sepharose CL-6B column. The enzyme was eventually eluted with a pulse of standard buffer supplemented with 5 mM NAD. SDS–PAGE analysis of the peak fractions revealed that the overexpressed recombinant enzyme was resolved from other contaminating proteins as a single protein band ofca. 50 kDa (Fig. 2A, lane d), which is in agreement with the protein sequence deduced from the putativeN. meningitidis gapNgene.
Eventually, recombinant bacterial GAPN was purified to electrophoretic homogeneity from the soluble protein
fraction fromE. coli(pNeis2) using a procedure modified from the previously reported for the archaeal GAPN from T. tenax [17]. Since dye-affinity chromatography was a very effective and straightforward purification step, no additional purification was required to obtain homogeneous preparations of the bacterial enzyme. Table2 summarizes a representative purification protocol. Values of ca.
26 U mg–1 of protein were obtained for the purified en- zyme with a yield of 20% and a purification factor ofca.
50 fold. SDS-PAGE analysis of the different fractions obtained during the purification procedure showed a pro- gressive enrichment of the 50-kDa protein band (Fig.2A), which was the only band present in the electrophoretically homogeneous final enzyme preparations (Fig.2A, lane d).
A polyclonal antibody raised against the purified recombinantN. meningitidisGAPN was used for Western blot analysis. This antibody clearly recognized a single polypeptide band of 50 kDa corresponding to the GAPN in all fractions obtained through the purification procedure (Fig.2B).
The purified enzyme was assayed at different tempera- tures to establish optimal activity conditions. Noteworthy, as is shown in Fig. 3A, theN. meningitidisenzyme exhibits maximum activity at 60–65C. It is interesting to note at this respect that the NAD-dependent GAPN of the ther- mophilic archaeon T. tenax showed highest activity at Fig. 2 Purification ofN. menigitidisGAPN from E. coliXL1-blue
cells transformed with pNeis2. (A) Coomassie blue-stained SDS- PAGE slab gel showing protein fractions of the different purification steps. Lane a, cell-free protein extract; lane b, 35–85% ammonium sulfate protein fraction; lane c, anion-exchange chromatography
eluate pool; lane d, Cibacron Blue Sepharose eluate pool (pure protein preparation). (B) Western blot analysis of the same fractions pictured in panel A using a monospecific antibody against the purified GAPN.
Aliquots ofca. 25lg of protein per lane were used. The arrow points to theca. 50-kDa GAPN subunit band
Table 2 Purification of recombinantNeisseria meningitidisstrain Z2491 GAPN fromE. colitransformed with pNeis2
Fraction Total protein (mg) Total activity (U) Specific activity (U mg–1) Purification (fold) Yield (%)
Crude extract 4312 2242 0.52 1 100
Ammonium sulfate (35–85%) 1151 1715 1.49 2.9 76
DEAE-cellulose eluate 44 805 18.30 35.2 36
Cibacron Blue Sepharose eluate 19 477 25.10 48.3 21
70C, and remaining activity after 100 min at 100C was 30% [17]. The optimum pH for the purified enzyme activity was determined over the pH range 5–13 using different buffers (acetate, imidazole, Tris and carbonate/
bicarbonate) and aldehyde substrates (Fig.3B). In all cases, the enzyme was most active in the pH 9–10 range, with half-maximal activity at about pH 8 and pH 10.6.
Buffer composition has been described to affect the enzyme activity. In this way, the enzymatic activity with
D-G3P was about 60% less active at pH 8 in phosphate buffer than in Tricine buffer and this is in agreement with previous reports on phosphate inhibition of plant and algal GAPNs [11,35]. 10 mM beta-mercaptoethanol and 2 mM dithiothreitol (DTT), generally used in buffers for other GAPNs [13, 14, 17], strongly inhibited the recombinant N. meningitidis GAPN (data not shown). Cofactor speci- ficity was also analyzed, the purified protein exhibited high specific activity with NAD but undetectable activity with NADP employing D-G3P as well as other aldehydes as substrates (Table 3). The assays with NADP were per- formed with various concentrations of the enzyme and at different pH values (5, 6, 7, 8, 9 and 10). Thus, the N.
meningitidis enzyme is strictly specific for NAD and NADP cannot replace NAD, rather it acts as a strong competitive inhibitor (75% inhibition in the presence of 2 mM NADP). A number of metabolites were tested as possible effectors on Neisseria GAPN. NADPH was an inhibitor and caused approximately 1.5 fold increase in the Km and 1.2 fold decrease in the Vmax (data not shown).
Contrary to the GAPN ofT. tenax[19], NADH, glucose-1- phosphate, AMP, ADP, ATP and fructose-6-phosphate did not affect kinetic properties of Neisseria GAPN and no change in cofactor preference from NAD to NADP in the presence of these metabolic intermediates was detected.
On the other hand, recombinant GAPN also shows lower NAD-dependent dehydrogenase activity with an hydroxy- aldehyde (glyceraldehyde, 4.5 U mg–1) and an aliphatic aldehyde (acetaldehyde, 3.9 U mg–1) (Table 3). Although the Km values for N. meningitidisGAPN were higher for
D-G3P than for other aldehydes, the catalytic efficiency was on the same order of magnitude for all of them. In contrast, the Kmvalue for NAD withD-G3P was roughly half than with the other substrates. Overall the catalytic properties indicate that this protein acts efficiently on a broad spec- trum of aldehyde substrates (cf. Table 3). Concerning the native molecular mass of the purified recombinant GAPN, non-denaturing PAGE yielded a value of ca. 208 kDa.
Therefore, and considering, as stated above, a subunit molecular mass value of ca. 50 kDa obtained by SDS- Fig. 3 Effects of temperature (panelA) and assay pH (panelB) on
the purifiedN. meningitidesGAPN activity
Table 3 Catalytic parameters of purifiedN. meningitidisGAPN with three different aldehyde substrates
Catalytic parameter Aldehyde substrate
G3P Glyceraldehyde Acetaldehyde
NAD-dependent specific activity (U mg–1) 25.10 ± 0.53 4.46 ± 0.25 3.91 ± 0.16
NADP-dependent specific activity (U mg–1) 0 0 0
Km(mM) 1.45 ± 0.10 0.20 ± 0.01 0.31 ± 0.06
Kcat(s–1) 24.51 ± 0.54 4.35 ± 0.10 4.03 ± 0.09
Kcat/Km(s–1mM–1) 16.90 21.75 13.00
Kmfor NAD (mM) 0.057 ± 0.005 0.088 ± 0.006 0.109 ± 0.011
Values are means (± standard errors) of three independent determinations
PAGE (Fig.2A, lane d), the nativeN. meningitidisGAPN should have an homotetrameric structure like eukaryotic and other bacterial GAPNs [13,14,17]. Isoelectric focus- ing on polyacrylamide gel of the N. meningitidis protein showed a single protein band focused at pH 6.3 (the esti- mated pIfor the native enzyme). This result suggests that the N. meningitidis enzyme consists of a single slightly- acidic isoform.
Comparative molecular phylogeny of GAPNs and other ALDHs
The peculiar kinetic properties of N. meningitidis GAPN prompted us to carry out molecular phylogenetic studies to clarify possible relationships with other members of the ALDH protein superfamily. A distance phylogenetic tree, constructed using a Clustal X-generated multiple sequence alignment and the distance (neighbor-joining) method [36], is shown in Fig.4. Maximum likelihood method was also employed giving similar results (data not shown). The tree was performed with various representatives of the ALDH superfamily and showed phylogenetic relationships among the ALDHs and GAPNs of the archaeal, eukaryotic and
prokaryotic groups, including new putative GAPNs present in databases and the novel glyceraldehyde dehydrogenase subclass recently found in archaea [37,38]. As can be seen in Fig. 4, archetypical non-phosphorylating NADP-depen- dent GAPDH proteins of Gram-positive bacteria and pho- tosynthetic eukaryotes form a compact group. The NAD- dependent GAPN ofT. tenaxis associated to the archaeal GAPN in a cluster distant from archetypical GAPN of plants and the Gram-positive bacteria. Interestingly, the Neisseria protein clustered with archaeal glyceraldehyde dehydrogenases (GADHs), although the later are strictly NADP-dependent enzymes with distinct functional and catalytic properties. The Neisseria GAPN has an overall identity of ca. 40% (ca. 60% of similarity) with the GADHs of the same cluster (Fig. 4). Thus, the archaeal GADHs showed no activity with NAD or aliphatyc alde- hydes and exhibited a marked activation by sulphydryl reagents, in contrast to the data reported in this work for the bacterial enzyme. It has also been suggested that the archaeal GADHs would function in a non-phosphorylated Etner–Doudoroff pathway [37]. Due to these marked differences we propose that the Gram-negative bacterial enzyme described in this work constitutes the first repre-
1 .
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s u m s e d e n e c S
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Fig. 4 Unrooted phylogenetic tree of ALDHs, glyceraldehyde dehydrogenases (GADH) and GAPNs from bacteria, archaea and photosynthetic eukaryotes, including the NAD-dependent protein of Neisseria meningitidis. This distance tree was constructed by the Neighbor-Joining algorithm from a CLUSTAL X multiple sequence alignment. Accession numbers of the protein sequences used are listed in the Material and Methods section. Biochemically character-
ized proteins are shown in bold and underlined. Numbers at nodes indicate statistical support (bootstrap values of 1,000 replicates) of selected groups. Note that N. meningitides GAPN clusters with archaeal GADHs, and appears closely related to archaeal GAPN orthologs, between ALDHs and archetypical bacterial/eukaryotic GAPNs assemblies
sentative of a novel aldehyde dehydrogenase subclass that may be involved in a number of carbon metabolic pathways not yet characterized. According to [7], ALDHs should present a high sequence similarity with the GAPN ancestor. Since theNeisseriaGAPN was located between the cluster of ALDHs and archaeal GAPN group we propose that the later and the bacterial protein could be primitive GAPN ortologues, although the Neisseria enzyme is more closely related to archaeal GADHs. These phylogenetic relationships may be due to horizontal gene transfers such as those proposed for some GAPDHs [3,7, 39] or alternatively theN. meningitidis GAPN could be a relic from ancestral Gram-negative bacteria where this enzyme could had been present.
A NAD/NADP-dependent GAPN had been previously described and biochemically characterized from the hy- perthermophilic crenarchaeoteT. tenax. This protein was proposed to work exclusively in carbohydrate oxidative pathways. The gapN gene encoding this T. tenax GAPN was cloned, and its protein product characterized and crystallized [17,19,40,41]. Recently, the allosteric effect of different activator molecules on the piridine nucleotide specificity of theT. tenax GAPN was studied [19]. These studies reflect, in the enzymatic activity, an important inclination in the specificity of the cofactor from NAD to NADP in the presence of activators. On the contrary, NeisseriaGAPN protein showed no such allosteric effect in the presence of these potential effectors, namely glucose-1- phosphate, AMP, ADP and fructose-6-phosphate. Full protein sequence alignments of GAPNs as well as other ALDHs showed that only GAPNs from hyperthermophilic archaea (T. tenax, Sulfolobus tokodaii, Sulfolobus solfa- taricus,Aeropyrum pernix andPyrococcus furiosus) have the conserved activator-binding site [19]. In accordance with this, full sequence alignments of archaeal GADHs or Neisseria GAPN with T. tenax GAPN showed only an overall 25–29% identity and no conservation of most of the binding site residues (data not shown). This may explain why Neisseria GAPN does not respond to the activators as the archaeal protein does.
In conclusion, the results presented in this paper are the first report on the cloning, purification and characterization of a bacterial NAD-dependent GAPN. This enzyme is different to archetypical bacterial GAPNs that present a strictly NADP-dependent aldehyde dehydrogenase activity (EC 1.2.1.9), which so far had been exclusively found in Gram-positive strains [15]. Previous studies demonstrated that some strains ofS. mutanscan use alternative mecha- nisms to generate NADPH for reductive biosynthetic reactions. NADP-specific GAPN is thought to be respon- sible for generating the NADPH needed for biosynthetic purposes [42]. It would be interesting to know the role of a NAD-generating enzyme in this context. Therefore, we are
currently investigating the molecular mechanism of N. meningitidis GAPN as well as the possible role played by this enzyme in the metabolism of this bacterium.
Acknowledgments The authors thank Prof. M. Losada for encouragement and help. This work is part of several collaborative Research Projects between the CNR (Morocco) and CSIC (Spain). It was supported by AECI (Spain) and collaborative grants of the Andalusian Government—Ministe`re d’Education et de la Recherche Scientifique of Morocco (Junta de Andalucı´a, Proyectos de Cooper- acio´n al Desarrollo en el A´ mbito Universitario de la Agencia And- aluza de Cooperacio´n Internacional A52/02, and A54/04), group PAI CVI-261 and grants from CNRST (Morocco).
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