Biotechnology Group, School of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India
Correspondence
Narayan S. Punekar
nsp{at}iitb.ac.in
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ABSTRACT |
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Details of the gel-filtration results and inhibitor studies are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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Fungal ammonium assimilation occurs via its incorporation into glutamate and glutamine. Some progress has been made in understanding the integrated regulation of the carbonnitrogen interface of metabolism (Mora, 1990; Marzluf, 1997
; Cooper, 2004
). Besides the glutamine synthetase/glutamate synthase route, the reaction catalysed by NADP-GDH (EC 1 . 4 . 1 . 4) mediates the reductive amination of 2-oxoglutarate to form glutamate. This places NADP-GDH at the crossroads of carbon and nitrogen metabolism, making this enzyme a candidate for metabolic control. It is argued that co-ordinated regulation of the two NADP-GDH isozymes in Saccharomyces cerevisiae enables a balanced utilization of 2-oxoglutarate for glutamate synthesis during diauxic growth (DeLuna et al., 2001
). Although transcription of the aspergillus NADP-GDH gene responds to carbon and nitrogen availability (Hawkins et al., 1989
; Cardoza et al., 1998
), knowledge of its regulation at the level of enzyme activity is lacking. Factors that govern NADP-GDH activity are important inputs in engineering carbon metabolic flux at this branch point.
GDHs from a variety of organisms have received considerable attention (Hudson & Daniel, 1993), but the biochemical characterization of NADP-GDH from filamentous fungi is limited (Blumenthal & Smith, 1973
; Botton & Msatef, 1983
; Martin et al., 1983
; Baars et al., 1995
; Bogati et al., 1996
). So far, only yeast NADP-GDHs are known to show a co-operative response towards 2-oxoglutarate (Holmes et al., 1989
; Perysinakis et al., 1994
; DeLuna et al., 2001
). As a step towards understanding the role of NADP-GDH in metabolic regulation at the carbonnitrogen interface we have (i) devised an effective method to purify this enzyme from aspergilli and (ii) identified three NADP-GDH inhibitors that are potentially of value in metabolic studies. On biochemical characterization, the A. niger NADP-GDH exhibited sigmoid saturation kinetics with 2-oxoglutarate, suggesting its importance in carbon flux distribution between the TCA cycle and nitrogen metabolism.
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METHODS |
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Chemicals required for gel electrophoresis and Sephadex G-25, Sephadex G-200, Sephacryl S-200, Sepharose CL-2B, Sepharose CL-4B and diethylaminoethyl (DEAE)-Sephacel were purchased from USB Chemicals and Amersham Biosciences. All other chemicals and media components used were of analytical reagent grade/of highest purity, locally available from Merck and other local suppliers.
2-Methyleneglutarate synthesis was accomplished by hydrolysis of 2-methylene-glutaronitrile (obtained from Sigma). 2-Methyleneglutaronitrile was heated in aqueous ethanol containing NaOH. The product was acidified with H2SO4 and extracted in ethyl acetate. 2-Methyleneglutarate, recrystallized from ethyl acetate, was characterized by its melting point (129130 °C) and by TLC on silica gel (80 % ethyl acetate in petroleum ether). Finally, the identity of 2-methyleneglutarate was established by elemental analysis and proton NMR.
Strains and growth conditions.
All aspergillus cultures, namely A. niger NCIM 565 (citric acid producer), Aspergillus oryzae NCIM 553, Aspergillus terreus NCIM 656, Aspergillus awamori NCIM 1225 and Aspergillus nidulans NCIM 1211, were obtained from National Chemical Laboratories, Pune, India, and stock cultures were maintained on potato dextrose agar (PDA) plates. The inoculum for liquid cultures was generated using spores from such PDA plates. The spore suspension was prepared in a sterile solution containing 0·005 % Tween 80. All species of aspergilli were grown on normal minimal medium (Kumar et al., 2000), except that A. terreus medium contained a higher concentration (2·0 %) of glucose. Fungal mycelia for NADP-GDH purification or activity assay were routinely grown on medium containing sodium nitrate (50 mM) as the sole source of nitrogen. The fungal strains were grown as a surface culture in 1 l Erlenmeyer flasks containing 100 ml of the appropriate medium at 37 °C for 30 h (maximal growth phase). However, A. terreus was grown on a shaker at 30 °C for 34 h. The mycelia were stored at 20 °C until use.
Yeast strains, namely S. cerevisiae Y05499 (BY4741; MATa his3 leu2
met15
0 ura3
0 YPR030w : : kanMX4), S. cerevisiae Y10345 (BY4739; MAT
leu2
0 lys2
0 ura3
0 YAL062w : : kanMX4) and S. cerevisiae Y11672 (BY4742; MAT
his3
1 leu2
0 lys2
0 ura3
0 YOR375c : : kanMX4), were obtained from Euroscarf. While strain Y05499 possessed both the Gdh isozymes, Y10345 and Y11672 were deleted in GDH3 and GDH1, respectively. All three strains were grown on yeast minimal medium (DeLuna et al., 2001
).
Preparation of affinity matrices.
The reactive dyes Cibacron red LS-B, Cibacron blue F3G-A, Procion yellow H-E3G and Procion red were coupled directly to various matrices (Sepharose CL-2B, Sepharose CL-4B, Sephacryl S-200 and Sephadex G-200) by a standard procedure (Bohme et al., 1972). The dye-coupled matrices were washed sequentially and extensively with double-distilled water, 3·0 M NaCl and buffers of pH 8·0 (0·1 M Tris/HCl) and pH 4·5 (0·1 M sodium acetate) to remove the unbound dye.
Enzyme extraction.
All enzyme extraction and purification steps were performed at 4 °C. Frozen fungal mycelia were crushed with an equal amount (w/w) of acid-washed sand in a pre-cooled mortar and pestle, for about 1520 min. The slurry was extracted with 5 vols (mycelial wet weight : volume) of ice-cold NADP-GDH extraction buffer, containing 100 mM potassium phosphate, 1·0 mM 2-mercaptoethanol, 1·0 mM EDTA and 1·0 mM PMSF (freshly added from a 100 mM stock in 2-propanol) at a final pH of 7·5. The extract was passed through two layers of cheesecloth and the filtrate was centrifuged at 15 000 g for 20 min in a refrigerated centrifuge. The final supernatant was designated and used as the crude enzyme extract.
Purification of NADP-GDH from A. niger and A. nidulans
Identical purification protocols were employed for the isolation of NADP-GDH from A. nidulans and A. niger. A typical procedure for the A. niger enzyme is described below.
Ammonium sulfate fractionation.
The enzyme was extracted as described above. The protein fraction precipitating between 35 and 70 % saturation of ammonium sulfate was collected.
Dye-affinity chromatography.
The active ammonium sulfate pellet (3570 % fraction) of the crude extract was dissolved in Buffer A (containing 20 mM potassium phosphate, 4·0 mM 2-mercaptoethanol and 1·0 mM EDTA at a final pH of 7·5) and loaded onto Cibacron red LS-B Sephadex G-200 (100 ml) matrix in a beaker. The affinity matrix was incubated with the protein for 3 h at 4 °C, with occasional swirling. The affinity matrix was conveniently used in a batch bindingelution mode. At each step of binding/washing/elution, the affinity matrix was separated from the supernatant by gentle centrifugation (3000 g for 10 min at 4 °C). The affinity matrix was then sequentially washed with Buffer A followed by 50 mM KCl in the same buffer. Elution of bound proteins was achieved with 0·2 M KCl in three batches of 50 ml each. The enzyme thus eluted from the affinity matrix was then saturated to 80 % with ammonium sulfate to precipitate the enzyme protein.
DEAE-Sephacel chromatography.
The enzyme eluted from the dye-affinity matrix was subsequently resuspended in Buffer A (10x dilution) and loaded on to a DEAE-Sephacel column (bed volume 20 ml) at a flow rate of 15 ml h1. Bound proteins were eluted at a flow rate of 18 ml h1, using a gradient of 00·5 M KCl (total volume 160 ml), and collected as 3 ml fractions. The fractions were assayed for enzyme activity and protein content.
Molecular mass determination.
The native molecular mass of A. niger NADP-GDH was determined on a Superdex G-75 prep-grade gel-filtration column (bed volume 320 ml) equilibrated with Buffer A. The column was calibrated with molecular mass standards (66443 kDa) from Sigma. The dye-affinity matrix-enriched enzyme was resuspended in the same buffer, loaded onto the column and eluted at a flow rate of 120 ml h1. Molecular mass was determined from a plot of log(molecular mass) against elution volume.
The subunit molecular mass of A. niger NADP-GDH was determined by SDS-PAGE with molecular mass standards (29205 kDa) from Sigma. MALDI-TOF analysis was performed on a Kratos PC Axima CFR machine.
Electrophoretic procedures and activity staining.
Both SDS-PAGE and native PAGE were performed with 7·5 % gels. Proteins on polyacrylamide gels were visualized with Coomassie blue R-250. Activity staining for NADP-GDH was performed according to established methods (Gabriel & Gersten, 1992) but with some modifications. After electrophoresis (native PAGE run at 4 °C), the gels were transferred to clean Petri dishes. In order to ensure uniform pH along their entire length, gels were immersed in ice-cold 100 mM Tris/HCl, pH 7·5, for 15 min prior to staining. These pH-equilibrated gels were immersed in the staining solution and incubated at room temperature. The staining solution (10 ml) contained 100 mM L-glutamate (1·0 ml), 0·4 mM NADP+ (0·5 ml), 100 mM Tris/HCl (pH 7·5, 2·0 ml), 1·0 mg nitroblue tetrazolium ml1 (2·5 ml), 1·0 mg phenazine methosulfate ml1 (2·5 ml) and distilled water (1·5 ml). For substrate controls, L-glutamate was omitted from the staining solution. During the entire process, the gels were protected from light. After incubation, the gels were washed sequentially in double-distilled water and 7·5 % acetic acid.
Immunochemical procedures.
Preimmune serum was collected before immunizing a white male New Zealand rabbit. Immunization was initiated by injecting 270 µg purified A. niger NADP-GDH protein emulsified in Freund's complete adjuvant. A second injection of 270 µg protein mixed with Freund's incomplete adjuvant was given a week later followed by two more injections (130 µg protein each time) at weekly intervals. Bleedings were taken from the marginal ear vein after 1 month and the serum obtained was decomplemented.
Proteins resolved on native PAGE were electroblotted onto nylon membrane (Hybond-N+) at constant voltage (200 V) for 34 h. After transfer, the membrane was blocked with blocking solution (1·5 % casein and 1·5 % gelatin in 50 mM phosphate buffer, pH 7·2) for 23 h. Subsequently, the antiserum (1 : 100 dilution) was added and the membrane incubated for 1 h. After adequate washing, the nylon membrane was incubated for 1 h with rabbit IgG antiserum (1 : 10 000) labelled with alkaline phosphatase. Immunoreactive bands were visualized by incubation with the substrate.
Enzyme assays and protein determination.
All enzyme activity measurements were made at the ambient temperature of 28 °C. NADP-GDH activity was measured by following the change in absorbance at 340 nm. Unless otherwise mentioned, the following standard assay conditions were used. Reductive amination was determined with an appropriate amount of enzyme protein in a reaction mixture (1·0 ml) containing 100 mM Tris/HCl, pH 8·0, 10 mM NH4Cl, 10 mM 2-oxoglutarate and 0·1 mM NADPH. The reaction was routinely started by the addition of NADPH. Oxidative deamination activity was determined with an appropriate amount of enzyme protein in a reaction mixture (1·0 ml) containing 100 mM Tris/HCl, pH 9·3, 0·4 mM NADP+ and 100 mM L-glutamate. This reaction was started by the addition of NADP+.
One unit of activity is defined as the amount of enzyme required to reduce/oxidize 1 µmol NADP+/NADPH min1. Specific activity is defined as U (mg protein)1. Protein was estimated (Bradford, 1976) using BSA as a reference.
Procedures for determination of NADP-GDH kinetics.
Pure A. niger NADP-GDH was used for the kinetic analysis. In all the kinetic measurements, sufficient enzyme was added to give a change in A340 of 0·10·2 min1. The reaction was carried out for 23 min and rates were obtained from the initial linear portion of the curves. Initiating the forward reaction of the A. niger NADP-GDH with different components of the assay resulted in different initial velocities. The initial velocities were always higher when the A. niger enzyme was exposed to either NADPH or NADPH plus 2-oxoglutarate before starting the reaction. In the reverse reaction, however, the initial velocity was the same irrespective of how the reaction was initiated. To overcome complications due to these variations, all enzyme kinetic assays were initiated by the addition of either NADPH (forward) or NADP+ (reverse). In substrate saturation experiments, standard assay conditions were employed except for the concentration of the substrate being varied. These changes are mentioned at appropriate places.
The various potential inhibitors were incubated with the enzyme in the standard forward assay and the reaction was subsequently initiated by the addition of NADPH. For experiments in which pH was varied, 25 mM acetic acid, 25 mM MES, 50 mM Tris was used as buffer to minimize the change of ionic strength with pH (Ellis & Morrison, 1982).
Statistical treatment and analysis of data.
Enzyme kinetic data presented are typical reproductions of at least three independent experiments and the data were analysed and plotted using the enzyme kinetics software SigmaPlot (version 8.0). A correlation coefficient (r) of 0·95 or higher was always obtained for these data. Experimentally determined values are presented as points, while the lines represent best-fits of these data.
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RESULTS |
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Purification of NADP-GDH from aspergilli
Several reactive dyes are reported to be useful in the purification of GDHs (Hudson & Daniel, 1993). The A. niger NADP-GDH activity was inhibited by the four reactive dyes tested, Cibacron red LS-B, Cibacron blue F3G-A, Procion yellow H-E3G and Procion red. Cibacron red LS-B inhibited the enzyme maximally and the enzyme bound most efficiently to Cibacron red LS-B-coupled Sephadex G-200 matrix. Therefore, Cibacron red LS-B was also coupled to Sepharose CL-2B, Sepharose CL-4B and Sephacryl S-200 using standard methods (Bohme et al., 1972
). All these matrices bound the A. niger NADP-GDH and the enzyme activity eluted efficiently using a KCl gradient (not shown). The Cibacron red LS-B dye-affinity purification (with a step KCl gradient elution) enriched the A. niger NADP-GDH by more than sevenfold and provided an almost homogeneous enzyme. However, a few minor contaminating protein bands were still found in this preparation. A subsequent DEAE-Sephacel gradient purification (Fig. 1a
) yielded electrophoretically homogeneous protein (Fig. 1b
) with a recovery of over 30 %. Identity of the purified protein as A. niger NADP-GDH was confirmed by activity staining (Fig. 1c
). The results of the entire purification protocol for obtaining pure A. niger NADP-GDH are summarized in Table 1
.
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Molecular properties.
The pure A. niger NADP-GDH retained full activity at 4 °C for a week, while the A. nidulans enzyme lost more than 50 % activity within 3 days. The UV spectrum of the homogeneous A. niger NADP-GDH protein showed a strong shoulder around 280 nm and a 1·0 mg ml1 solution of the pure enzyme protein gave an A280 of 1·45. The native molecular mass of pure A. niger enzyme was determined to be 350 kDa by FPLC on a calibrated Superdex G-75 prep-grade gel-filtration column. Details of the gel-filtration results are available as Supplementary Fig. S1 with the online version of this paper. The subunit molecular mass of A. niger NADP-GDH as determined by SDS-PAGE was 50 kDa. On MALDI-TOF analysis, the pure protein showed a single sharp peak with Mr 49 153. From a comparison of its native and subunit molecular masses, the A. niger NADP-GDH appeared to be a hexamer. Sequencing attempts suggested that the protein had a blocked N terminus.
Polyclonal antibodies raised against the purified A. niger NADP-GDH were used to immunoblot NADP-GDHs from other aspergilli, namely A. nidulans, A. oryzae and A. terreus, and three S. cerevisiae strains [Y05449, Y10345 (GDH3) and Y11672 (GDH1
)]. Cross-reacting protein bands were observed on Western blots in lanes corresponding to A. niger, A. nidulans, A. oryzae and A. terreus crude extracts (Fig. 3
). However the antiserum did not cross-react with either the Gdh1 or Gdh3 protein of S. cerevisiae.
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2-Oxoglutarate saturation and co-operativity
The pure A. niger NADP-GDH showed sigmoidal saturation behaviour with respect to 2-oxoglutarate (Fig. 4). These saturation data, when fitted to the Hill equation, gave S0·5 and nH values of 4·8 mM and 2·50, respectively (Fig. 4a, b
). When tested, the purified enzyme from A. nidulans also showed co-operative 2-oxoglutarate saturation kinetics, albeit with a lower nH value of 1·5 (Fig. 4c, d
). It was of interest to explore whether the observed co-operative behaviour was (i) an artefact of purification and (ii) a common feature of other aspergilli. The NADP-GDH in the crude mycelial extracts of four different aspergilli showed sigmoid 2-oxoglutarate kinetics (Fig. 5
). However, their individual nH values were different.
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Reductive amination assays for A. niger NADP-GDH were invariably initiated by the addition of NADPH (see Methods). The enzyme activity was stimulated by prior incubation with NADP+ (the product), especially at lower 2-oxoglutarate concentrations. On further analysis, NADP+ showed a biphasic effect; while it stimulated the enzyme at lower 2-oxoglutarate levels, the activity was slightly inhibited at higher concentrations (Fig. 6).
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DISCUSSION |
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A. niger mycelia grown on nitrate as the sole source of nitrogen exhibited maximum NADP-GDH specific activity, a feature common to other aspergilli (Cardoza et al., 1998; Pedersen et al., 1999
). Nitrate-grown A. niger mycelia were therefore suitable starting material in the purification of NADP-GDH activity. Dye-affinity chromatography has been exploited to purify NADP-GDH from a few organisms (Watson et al., 1978
; Agrawal & Rao, 1983
; Aguirre & Hansberg, 1988
; Syed et al., 1991
). Keeping this in mind, four different reactive dyes were explored and the Cibacron red LS-B-coupled matrix was most effective in purifying the A. niger NADP-GDH (Fig. 1b
). An added advantage of this novel dye-affinity purification is a simple and convenient elution by KCl, while, in all the earlier methods, NADP(H) was required for elution. It is thus possible to obtain milligram quantities (Table 1
) of electrophoretically pure A. niger NADP-GDH. The general applicability of this procedure to NADP-GDH from other aspergilli was obvious, as we could purify the A. nidulans enzyme to homogeneity (Fig. 2
and Table 1
) by the same method.
Unlike the NADP-GDH from Neurospora crassa and Aspergillus ochraceus (Blumenthal & Smith, 1973; Agrawal & Rao, 1983
), the A. niger NADP-GDH was unusually stable. In contrast, the A. nidulans enzyme was very labile, leading to poor recoveries during purification (Table 1
). This loss could not be attributed to proteolytic degradation, since the stored (1-week-old) inactive protein moved as a single band on native PAGE. Based on native and subunit molecular masses, the A. niger enzyme appeared to be a hexamer, like the other NADP-GDHs reported (Blumenthal & Smith, 1973
; Botton & Msatef, 1983
; Martin et al., 1983
; Hudson & Daniel, 1993
; Baars et al., 1995
; Bogati et al., 1996
). Since its N terminus appears to be blocked, the ready availability of milligram quantities of the A. niger protein should now permit access to internal peptide fragments for sequencing. The only report of a partial protein sequence is for the Penicillium chrysogenum NADP-GDH (Bogati et al., 1996
; Diez et al., 1999
). While S. cerevisiae contains two distinct NADP-GDH genes, filamentous fungi including N. crassa, P. chrysogenum and various aspergilli contain a single copy of the NADP-GDH gene (DeLuna et al., 2001
). We compared the known fungal NADP-GDH sequences (deduced amino acid sequences) using CLUSTAL W. Among filamentous fungi, NADP-GDHs from A. awamori (Cardoza et al., 1998
), A. nidulans (Hawkins et al., 1989
) and Aspergillus fumigatus (http://www.tigr.org) form a cluster. Pairwise identities (generated using BLAST) also indicated greater than 80 % identity between these three deduced protein sequences. While it would be useful to have the A. niger NADP-GDH protein sequence in hand, antibodies provide yet another tool for comparison. NADP-GDH proteins from all the aspergilli tested cross-reacted with the antiserum raised against pure A. niger NADP-GDH (Fig. 3
), indicating their antigenic relatedness and similarity. The yeast isozymes (DeLuna et al., 2001
) are clearly distinct, as they did not cross-react with these antibodies.
The biosynthetic role of fungal NADP-GDH is well supported by genetic (Fincham, 1962; Kinghorn & Pateman, 1973
; Fincham et al., 2000
), physiological (Martin et al., 1988
; Kusnan et al., 1989
; Schwartz et al., 1991
) and kinetic (Hudson & Daniel, 1993
) data. The kinetic constants obtained for the various substrates of the A. niger NADP-GDH (Table 2
) are consistent with its role in ammonium assimilation. Ammonium could also act as a product inhibitor in the reverse reaction (Baars et al., 1995
). A strong inhibition of the reverse reaction by ammonium (Ki of 0·40 mM) and a substantial difference in the A. niger NADP-GDH affinities for ammonium and L-glutamate (as reflected by their respective Km values) favour the enzyme reaction towards glutamate formation in vivo. Absence of inhibition of the reductive amination reaction by glutamate (>100 mM; not shown) is also consistent with its assimilatory role.
There is a paucity of information on NADP-GDH inhibitors, with the literature being biased towards NAD-GDH (Caughey et al., 1956; Veronese et al., 1974
; Stevens et al., 1989
). Attempts to generate more isophthalate derivatives as inhibitors and also to obtain useful conformationally restricted substrate analogues for GDHs have been made (Cunliffe et al., 1983
; Denton et al., 2001
). Specific NADP-GDH inhibitors, in addition to dissecting metabolic details (Kusnan et al., 1987
), could be valuable in mechanistic studies. Selected NAD-GDH inhibitors were included in our screening (Table 3
) on two counts: (i) the extensive similarity in chemical mechanism of the NAD-GDH and NADP-GDH reactions (Srinivasan et al., 1988
; Hudson & Daniel, 1993
; Brunhuber & Blanchard, 1994
) and (ii) the largely identical active-site geometry for the two reactions and conserved structural layout of their active-site amino acid residues (Baker et al., 1992
). This is the first report of NADP-GDH inhibition by isophthalate (the most studied NAD-GDH inhibitor) and identifies 2-methyleneglutarate as an equally potent non-aromatic inhibitor.
Considering the proposed iminoglutarate intermediate (Fig. 7; structure 3) and the observed potent isophthalate inhibition, other dicarboxylate compounds were evaluated for their inhibitory potential. As expected, inhibition of A. niger NADP-GDH by the three compounds (isophthalate, 2,4-pyridinedicarboxylate and 2-methyleneglutarate) was competitive with respect to L-glutamate (Supplementary Fig. S2). Preliminary data (unpublished) suggest their promise as in vivo inhibitors of A. niger NADP-GDH and fungal nitrogen metabolism. 2-Methyleneglutarate, owing to its close structural resemblance to 2-oxoglutarate and iminoglutarate (Srinivasan et al., 1988
; Fisher et al., 2001
), was synthesized and tested as a potential NADP-GDH inhibitor. Although not a substrate, this molecule was an excellent inhibitor of the NADP-GDH reaction. Itaconate, a lower homologue of 2-methyleneglutarate, unexpectedly activated A. niger NADP-GDH, and this needs further investigation. Both 2,4-pyridinedicarboxylate and 3,5-pyrazoledicarboxylate were anticipated to mimic iminoglutarate; only the former showed significant inhibition of A. niger NADP-GDH (Supplementary Fig. S2 and Table 3
). As invoked in the case of isophthalate (Caughey et al., 1956
), the distance between the two negatively charged groups (an important determinant for binding) possibly renders 3,5-pyrazoledicarboxylate and the other pyridinedicarboxylate isomers less effective. An interesting proposition is that the nitrogen atom of 2,4-pyridinedicarboxylate, occupying a position similar to the one in iminoglutarate, may contribute to its potency. In fact, the synthesis and properties of related compounds (
1-piperidine-2,4-dicarboxylate; Fig. 7
, structure 8) have already been reported (Nielsen et al., 2001
) and are worth pursuing.
The homogeneous A. niger NADP-GDH exhibited a strong sigmoidal response (nH of 2·5) towards varying concentrations of 2-oxoglutarate (Fig. 4). The A. nidulans enzyme also showed co-operative behaviour, albeit to a lesser degree. The sigmoid saturation was not an artefact of purification, but an inherent property of aspergillus NADP-GDH, as crude mycelial extracts also showed similar behaviour. This novel report of an allosteric 2-oxoglutarate interaction with NADP-GDH appears to be a regulatory feature common at least to the four aspergilli tested. Among the fungal enzymes studied so far, only yeast NADP-GDHs are reported to exhibit sigmoidal 2-oxoglutarate saturation (Holmes et al., 1989
; Perysinakis et al., 1994
; DeLuna et al., 2001
). The two S. cerevisiae isozymes (Gdh1p and Gdh3p) are implicated in fine-tuning yeast glutamate synthesis and carbon metabolism during the diauxic shift. While both exhibited co-operative behaviour towards varying concentrations of 2-oxoglutarate, only Gdh3p showed a pH dependence of its Hill coefficient (DeLuna et al., 2001
). A. niger NADP-GDH resembles yeast Gdh1p in that the nH value (from 2-oxoglutarate saturation) was pH independent. The observed activation at lower 2-oxoglutarate concentrations by some of the dicarboxylic acids (Table 3
) and the biphasic response to NADP+ (Fig. 6
) may be manifestations of this sigmoidal behaviour of A. niger NADP-GDH. At subsaturating concentrations of 2-oxoglutarate (the substrate exhibiting sigmoid saturation), such ligands could stimulate the velocity by promoting the T
R allosteric transition (Gerhart & Pardee, 1963
; Segel, 1993
; MacRae et al., 2002
). They act as pure competitive inhibitors at higher concentrations. Co-operative effects of substrate and substrate analogue (succinate) on N. crassa NADP-GDH conformational states are documented (Fincham et al., 2000
).
Attempts to engineer microbial metabolic fluxes through manipulations at the level of NADP-GDH (Marx et al., 1999; Nissen et al., 2000
) clearly suggest the significance of this enzyme at the carbonnitrogen interface of metabolism. It is interesting that, although NADP-GDH is central to aspergillus nitrogen metabolism (and ammonia assimilation), its activity is amenable to regulation by 2-oxoglutarate. The co-operative kinetics with 2-oxoglutarate suggest that the A. niger enzyme is responsive to and has the potential to set the carbon flux at the interface of carbon and nitrogen metabolism. Spare carbon flux, available due to a TCA cycle block after 2-oxoglutarate during acidogenesis, may thus account for the increased flux towards glutamate (Meixner-Monori et al., 1985
; Kumar et al., 2000
; Ruijter et al., 2002
). Selective enzyme inhibitors are useful in mechanistic studies and are potential metabolic tools to achieve pathway blocks in vivo (Kusnan et al., 1987
, 1989
). The inhibitors identified and characterized in this study are expected to help to define the role of NADP-GDH further in fungal carbon metabolic flux at this branch point.
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ACKNOWLEDGEMENTS |
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Received 4 November 2004;
revised 12 January 2005;
accepted 17 January 2005.
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