Aspergillus terreus NADP-glutamate dehydrogenase is kinetically distinct from the allosteric enzyme of other Aspergilli

mycological research 113 (2009) 1121–1126

journal homepage: www.elsevier.com/locate/mycres

Aspergillus terreus NADP-glutamate dehydrogenase is kinetically distinct from the allosteric enzyme of other Aspergilli Rajarshi CHOUDHURY, N. S. PUNEKAR* Biotechnology Group, School of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Mumbai-400076, India

article info

abstract

Article history:

NADP-Glutamate dehydrogenase (NADP-GDH) located at the interface of carbon and nitro-

Received 22 April 2009

gen metabolism has the potential to dictate fungal carbon flux. NADP-GDH from Aspergillus

Accepted 14 July 2009

terreus, itaconate producer and an opportunistic pathogen, was purified to homogeneity us-

Available online 18 July 2009

ing novel reactive dye-affinity resins. The pure enzyme was extensively characterized for

Corresponding Editor:

its biochemical and kinetic properties and compared with its well studied Aspergillus niger

Daniel C. Eastwood

counterpart. The A. terreus NADP-GDH was more stable and showed non-competitive ammonium inhibition with respect to glutamate. It exhibited hyperbolic 2-oxoglutarate satu-

Keywords:

ration albeit with a weak substrate inhibition. This is in contrast to the allosteric nature of

Allostery

the enzyme from other Aspergilli. Differential susceptibility to chymotrypsin is also consis-

Aspergillus terreus

tent with the absence of substrate cooperativity and conformational changes associated

NADP-glutamate dehydrogenase

with A. terreus NADP-GDH. The non-allosteric nature of A. terreus NADP-GDH provides

2-oxoglutarate saturation

a unique opportunity to assess the contribution of allostery in metabolic regulation. ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction The genus Aspergillus comprises a well known group of industrially important filamentous fungi capable of producing organic acids (Ruijter et al. 2002) and extra-cellular enzymes (Bennett 1998). Fungal overflow of metabolism leading to citrate (Aspergillus niger) and itaconate (Aspergillus terreus) share many similarities (Wilke & Vorlop 2001). However the presence of cis-aconitate decarboxylase appears to be unique to A. terreus (Dwiarti et al. 2002; Kanamasa et al. 2008). Besides its industrial importance as itaconate and lovastatin (Manzoni & Rollini 2002) producer, A. terreus has attracted increasing attention as an opportunistic fungal pathogen. The role of Aspergillus nitrogen metabolism in pathogenesis is becoming apparent (Krappmann & Braus 2005). NADP-Glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) is a significant mediator

of ammonium assimilation in Aspergilli (Macheda et al. 1999; Choudhury et al. 2008). þ 2-Oxoglutarate þ NHþ 4 þ NADPH % L-Glutamate þ NADP

Biosynthetic NADP-GDH located at the crossroads of fungal carbon and nitrogen metabolism is a potential candidate for metabolic control. It is an important member of the fungal 4-aminobutyrate shunt (Santosh Kumar & Punekar 1997). This enzyme characterized from two different yeast species exhibits non-Michaelian 2-oxoglutarate saturation (DeLuna et al. 2001; Perysinakis et al. 1994). The two NADP-GDH isozymes from Saccharomyces cerevisiae are implicated in adjusting the carbon flux during diauxic shift (DeLuna et al. 2001). The enzyme from A. niger (as also from three other Aspergilli) displays allosteric interaction with 2-oxoglutarate (Noor & Punekar

* Corresponding author. Tel.: þ91 022 2576 7775; fax: þ91 022 2572 3480. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2009.07.009

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2005). This has a potential to set the carbon flux at the carbon– nitrogen interface. Preliminary studies with crude A. terreus extracts however showed unexpected hyperbolic 2-oxoglutarate saturation. The present study is an attempt to characterize this kinetic feature of A. terreus NADP-GDH. Towards this objective the A. terreus enzyme was purified to electrophoretic homogeneity through dye-affinity chromatography and its biochemical and kinetic properties compared with the A. niger counterpart. The hyperbolic 2-oxoglutarate saturation of A. terreus NADP-GDH provides an avenue to evaluate the contribution of allostery in carbon flux regulation.

Experimental Strains and growth conditions The fungal cultures namely, Aspergillus niger NCIM 565 and Aspergillus terreus NCIM 656 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 sterile, solution containing 0.005 % Tween 80. Mycelia were harvested after growth on normal minimal medium (Noor & Punekar 2005) except that A. terreus medium contained a higher concentration (2.0 %) of glucose and the initial pH was adjusted to 4.2. Fungal mycelia for NADP-GDH purification or activity assay were routinely grown as mentioned before (Noor & Punekar 2005).

Purification of NADP-GDH from A. terreus All enzyme extraction and purification steps were performed at 4  C. Aspergillus terreus ‘crude enzyme extract’ was obtained from frozen fungal mycelia essentially as reported for the Aspergillus niger NADP-GDH (Noor & Punekar 2005). The protein fraction precipitating between 35 % and 70 % saturation of ammonium sulfate was collected for further processing. The A. terreus NADP-GDH ammonium sulfate pellet was dissolved in 20 mM potassium phosphate buffer (containing 1.0 mM 2-mercaptoethanol and 1.0 mM EDTA; final pH of 7.5) and desalted on a Sephadex G-25 column using the same buffer. The desalted protein was loaded onto a 50 ml CibaFixAR-017 affinity resin (Ekkundi et al. 2006) at a flow rate of 10 ml h1. The column was washed with 3–5 column volumes of the above buffer and subsequently eluted with 0–0.8 M linear gradient of KCl. Peak enzyme fractions were collected, concentrated by ammonium sulfate precipitation and loaded on Red Sepharose column (Cibacron Red LS-B directly coupled to Sepharose CL-6B; 20 ml), at a flow rate of 15 ml h1 and washed with 5 column volumes of buffer. The bound enzyme was eluted with linear gradient of 0–0.8 M KCl (20 ml h1) and 3 ml fractions were collected. The A. niger NADP-GDH was also purified for comparative study, as reported (Noor & Punekar 2005) but with minor modifications. Cibacron Red LS-B linked through a spacer to Sepharose CL-6B was used for convenience of handling.

R. Choudhury, N. S. Punekar

NADP-GDH assays and kinetics 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 and the data analyzed as mentioned before (Choudhury & Punekar 2007). One unit of activity is defined as the amount of enzyme required to reduce/oxidize 1 mmol NADPþ/NADPH min1. Specific activity is defined as U (mg protein)1. Protein was estimated (Bradford 1976) using bovine serum albumin as a reference. In the substrate saturation experiments, the standard assay conditions were employed except for the concentration of the substrate being varied. These and other changes made to the assay are mentioned wherever appropriate. The various inhibitors were incubated with the enzyme in the standard forward/reverse assay and the reaction was subsequently initiated by the addition of NADPH or NADPþ, as required.

Proteolytic digestion and electrophoretic procedures Proteolysis of the purified NADP-GDH protein (from Aspergillus terreus and Aspergillus niger) was performed by incubation with either chymotrypsin or trypsin. Digestions (in 200 ml of 20 mM phosphate buffer, pH 7.5) were set up at 37  C in a water bath. For every 35–50 mg pure NADP-GDH protein 2.5 mg of sequencing grade protease was used. Aliquots (20 ml) of proteolysis mixture were taken at time intervals to assay for enzyme activity and electrophoresis. Protection by different ligands was also monitored in similar experiments. Pure NADP-GDH protein was pre-incubated with different ligands before the protease addition. Proteins were analyzed both on native PAGE (7.5 % gels) and SDS-PAGE (10 % gels). The protein bands were visualized by staining with Coomassie Blue R-250. The A. terreus protein was some what poorly stained than the A. niger NADP-GDH. Activity staining for NADP-GDH on native gels was performed as before (Noor & Punekar 2005).

Results and discussion Purification and characterization of A. terreus NADP-GDH Crude NADP-GDH preparations from Aspergillus terreus behaved kinetically unlike the enzyme from other Aspergilli. Since the sigmoid 2-oxoglutarate saturation of this enzyme

Table 1 – Summary of steps employed in NADP-GDH purification from Aspergillus terreus Steps

Crude extract 35–70 % Ammonium sulfate fraction CibaFixAR-017 (dye affinity) Red Sepharose (dye affinity)

Fold Percent Total Specific units activity purification recovery (U mg1) 218 125

0.9 2.3

1.0 2.6

100 57

113

5.7

6.2

52

105

17.6

19.1

48

Aspergillus terreus NADP-glutamate dehydrogenase is non-allosteric

Table 2 – A comparison of Michaelis constants of NADPGDH from Aspergillus terreus and Aspergillus niger Km (mM)

Substrate

a

A. terreus NADP-GDH A. niger NADP-GDH Ammonium 2-Oxoglutarate NADPH L-Glutamate NADPþ

0.95 6.0 0.055 24.1 0.045

1.05 4.78b 0.011 34.6 0.017

Activity (µmol/min/mg)

has consequences to the distribution of carbon flux between carbon and nitrogen metabolism, it was of interest to further define its unusual kinetic features. For this the purification of A. terreus NADP-GDH was undertaken. A protocol established

6

4

2

0

10

20

Table 3 – Substrate pre-incubation effect on the forward reaction of NADP-GDH Reaction started witha

A. niger NADP-GDHb

A. terreus NADP-GDHb

NADPH 2-Oxoglutarate NHþ 4 Enzyme

9.70 8.21 14.1 6.17

4.60 4.50 4.80 4.40

a Reaction started with addition of appropriate substrate or enzyme in standard reductive amination reaction and monitored (see Experimental). b All numbers are specific activities (mmol min1 mg1) for the forward reaction.

a Data from (Noor & Punekar 2005). b Sigmoidal behavior with nH ¼ 2.7; S0.5 reported.

0

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for other Aspergilli (Noor & Punekar 2005) was not successful; the A. terreus enzyme from crude extracts bound poorly to Cibacron Red affinity resin as a first step of purification. After screening a series of reactive dye coupled resins (Ekkundi et al. 2006), CibaFixAR-017 affinity matrix was selected. Substantial enrichment of the enzyme was achieved by this step alone. However a subsequent Red Sepharose column provided a homogeneous preparation. The two step purification resulted in 50 percent recovery with 20 fold enrichment. Table 1 summarizes the results of a successful purification strategy developed for the A. terreus enzyme. The differences in dye-affinity resin binding by the A. terreus NADP-GDH were also consistent with the observed inhibition characteristics using the corresponding free dyes (Novel dye-affinity matrix library for protein chromatography, proteomics and scale up; N.S. Punekar, CHI PepTalk conference on ‘‘Protein Purification & Recovery’’, Jan. 12–14, 2009, San Diego, USA).

30

2-Oxoglutarate (mM)

(µmol/min/mg)

-1

0.20

0.15

0.10

0.05

.04

-0.02

0.00

0.02

0.04

0.06

-1

Glutamate (mM)

Fig 1 – 2-Oxoglutarate saturation and ammonium inhibition kinetics of Aspergillus terreus NADP-GDH. The activity of pure enzyme with varying 2-oxoglutarate was monitored (top) in a standard reductive amination assay with NADPH (100 mM) and ammonium (10 mM) as the fixed substrates. The deamination activity was assayed in the presence of different fixed concentrations (None, C; 0.05 mM, B; 0.1 mM, ; and 0.3 mM, 7) of ammonium chloride and the double reciprocal plots for substrate saturation with L-glutamate is shown (bottom).

Fig 2 – Chymotrypsin cleavage of pure NADP-GDH from Aspergillus terreus and Aspergillus niger. Panel A shows native PAGE of pure A. terreus protein either stained for protein (left frame) or activity (right frame). A. terreus protein sampled after 60 min of incubation with (Lanes A2 and A4) and without (Lanes A1 and A3) chymotrypsin was separated on 7.5 % Native-PAGE. Panel B shows SDS-PAGE of A. terreus (Lane B2) and A. niger (Lane B4) NADP-GDHs after chymotryptic digest and proteolysed products are shown with arrows. Corresponding unproteolysed controls are marked in Lane B1 (A. terreus) and Lane B3 (A. niger). The standard molecular weight markers as shown in Panel B (include 97, 66, 43, 29, 20 and 14 kDa, respectively).

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R. Choudhury, N. S. Punekar

10 20 30 40 50 60 70 80 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| A. niger MSNLPHEPEFEQAYKELASTLENSTLFQKNPEYRKALAVVSVPERVIQFRVVWEDDAGNVQVNRGFRVQFNSALGPYKGG A. terreus .....V................................................N.K.E..................... Consensus ***** ************************************************:* *:********************* 90 100 110 120 130 140 150 160 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| A. niger LRFHPSVNLSILKFLGFEQIFKNALTGLNMGGGKGGSDFD PKGKSDNEIRRFCVSFMTELCKHIGADTDVPAGDIGVTGR A. terreus ..............................................S.......A......R.................. Consensus **********************************************.*******:******:****************** 180 190 200 210 220 230 240 170 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| A. niger EVGFLFGQYRKIRNQWEGVLTGKGGSWGGSLIRPEATGYGVVYYVEHMIAHATNGQESFKGKRVAISGSGNVAQYAALKV A. terreus .I.Y.......L..S........................................A...A.................... Consensus *:*:*******:**.**************************************** *** ******************** 250 260 270 280 290 300 310 320 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| A. niger IELGGSVVSLSDSQGSLIIN --GEGSFTPEEIELIAQTKVERKQLASIVGAAP FSDANKFKYIAGARPWVHVGKVDVALP A. terreus .....R............VKDTAK.....A..DA..AL..D...I.EL.SD.A. --.D..T.LP.Q.......A...... Consensus ***** ************:: .:***** **: ** **:***:*.:*. *.* *:**.*:.* ******* ****** 360 370 380 390 400 330 340 350 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| A. niger SATQNEISGEEAQVLINAGCKFIAEGSNMGCTQEAIDTFEAHRTANAGAAAIWYAPGKAANAGGVAVSGLEMAQNSARLS A. terreus ......V......A..A................A...A.....E..K................................. Consensus ******:******.** **************** ***:***** ** ********************************* 410 420 430 440 450 460 ....|....|....|....|....|....|....|....|....|....|....|....|.. A. niger WTSEEVDARLKDIMRDCFKNGLETAQEYATPAEGVLPSLVTGSNIAGFTKVAAAMKDQGDWW A. terreus ..A...........KS..Q...D..K.................................... Consensus **:***********:.**:*** :**:************************************ Fig 3 – ClustalW consensus for the NADP-GDH amino acid sequences from Aspergillus terreus and Aspergillus niger. Pair-wise sequence alignment was performed in BioEdit using ClustalW.

The purified A. terreus NADP-GDH had a native molecular mass of 350 kDa by FPLC on a calibrated Superdex G-75 column (not shown) and on SDS-PAGE shows a subunit molecular weight of 50 kDa (see below). Like its Aspergillus niger counterpart, the A. terreus enzyme appears to be hexameric (Noor & Punekar 2005). It showed an alkaline pH optima of 8.0 (reductive amination) and 9.5 (oxidative deamination) and specifically used NADP(H). A number of metabolites have been found to elicit inhibition or activation of glutamate dehydrogenases (Hudson & Daniel 1993; Bonete et al. 1996). The pure A. terreus enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzyme activity. Most TCA cycle related metabolites (namely citrate, cis-aconitate, isocitrate, succinate, malate, oxaloacetate and itaconate) did not affect the forward reaction at 1.0 mM. The enzyme activity was not affected by the presence of AMP or ADP (alone at 1.0 mM or in combination with 2.0 mM Mg2þ) whereas ATP, GDP and GTP (in the presence of 2.0 mM Mg2þ) slightly activated the biosynthetic reaction of this enzyme (30 % stimulation). The results suggest that these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of NADP-GDH from Aspergilli (Noor & Punekar 2005) including the A. terreus enzyme. However the A. terreus NADP-GDH was unusual in that its forward reaction rate was 80 % inhibited by 5 mM ZnCl2.

Unusual features of A. terreus NADP-GDH: 2-oxoglutarate saturation The purified NADP-GDH from Aspergillus terreus was assessed for its kinetic properties. Different substrate saturation curves were generated (not shown) for both the forward and reverse activity of this enzyme. Michaelis constants determined from these data are summarized in Table 2. The kinetic constants were comparable to the corresponding parameters of the well characterized Aspergillus niger enzyme. A significant difference however was the hyperbolic 2-oxoglutarate saturation of crude NADP-GDH from A. terreus mycelial extracts. The Michaelian 2-oxoglutarate saturation reproduced with the purified enzyme (Fig 1) indicated that this kinetic feature was inherent to the protein (and not a result of interaction with some component(s) in the crude extracts). The A. terreus NADP-GDH also differed from its A. niger counterpart in terms of inhibitor interactions. The enzyme was poorly inhibited by isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate (Choudhury & Punekar 2007). Although ammonium inhibited the oxidative deamination activity (as a product), this inhibition was non-competitive with respect to L-glutamate (Ki ¼ 0.17 mM and a ¼ 1.1; Fig 1). This feature makes ammonium a more effective inhibitor of the reverse reaction and emphasizes the anabolic role for this

Aspergillus terreus NADP-glutamate dehydrogenase is non-allosteric

enzyme. In similar experiments with NADPþ as the varied substrate, ammonium was a potent non-competitive inhibitor (Ki value of 0.18 mM and a ¼ 1.3; not shown). The sigmoid 2-oxoglutarate kinetics and allosteric transitions of A. niger NADP-GDH are well correlated with its biphasic response to NADPþ. This was also manifest as substrate pre-incubation effects on the reaction kinetics (Noor & Punekar 2005; Fincham et al. 2000). Such pre-incubation effects on the A. terreus NADP-GDH were therefore evaluated. The amination (forward) reaction was initiated in all four possible ways and the activity monitored. Unlike A. niger NADP-GDH, the A. terreus enzyme activity was not affected when pre-incubated with a combination of 2-oxoglutarate and NADPH (Table 3). Similar results were observed with crude mycelial enzyme preparations as well. A. terreus NADP-GDH thus does not display these ligand-dependent conformational transitions – a feature consistent with its non-allosteric nature of 2-oxoglutarate saturation. Ligand induced protein conformational changes could be followed through differential susceptibility to proteolysis. Effect of chymotrypsin and trypsin on A. terreus NADP-GDH was studied where the A. niger enzyme served as a control. Both enzyme activities were stable towards tryptic digestion (up to 2 h) and this stability was unaffected by the presence of different substrate combinations (not shown). The two enzymes were differently susceptible to chymotrypsin. A. terreus NADP-GDH remained fully active even after 2 h of incubation with chymotrypsin. However the A. niger enzyme lost 50 % activity within 30 min and a combination of 2-oxoglutarate and NADPþ afforded near complete protection against chymotrypsin. The proteolytic susceptibility of the two NADP-GDH activities was complemented by PAGE analysis. Both A. niger and A. terreus proteins were cleaved by chymotrypsin, albeit differently (Fig 2). The loss of A. niger NADP-GDH activity is accompanied by the appearance of a new 36 kDa protein band (Fig 2B). Formation of this proteolysis product was decreased when combination of 2-oxoglutarate and NADPþ was present (data not shown). In contrast to the A. niger enzyme, chymotryptic cleavage released a smaller 14 kDa fragment from A. terreus NADP-GDH (Fig 2B). Despite this limited proteolysis, the A. terreus enzyme was functional as seen by activity staining on native PAGE (Fig 2A). Differential susceptibility to proteolysis between the two Aspergillus enzymes is consistent with their distinct 2-oxoglutarate kinetics.

Conclusions Uniqueness of Aspergillus terreus enzyme among the NADPGDHs from Aspergilli was apparent from the initial 2-oxoglutarate saturation data and its interaction with dye-affinity matrices. This was further confirmed through results presented in this study. Purified enzyme exhibited hyperbolic 2-oxoglutarate saturation and the substrate interaction was clearly not sigmoid. Being a member of the fungal 4-aminobutyrate shunt (Santosh Kumar & Punekar 1997), this feature of A. terreus NADP-GDH assumes significance in influencing the distribution of carbon flux at 2-oxoglutarate node. However, with both the hyperbolic (A. terreus) and allosteric (Aspergillus niger) NADP-GDHs in hand, regulatory significance of this

1125

kinetic feature is now amenable to study. We have compared the all the eight available Aspergillus NADP-GDH sequences (deduced amino acid sequences). Pair-wise identities (BLASTP) for A. terreus NADP-GDH (http://www.broad.mit.edu) with that of A. niger protein indicated a very high degree of conservation (460 amino acid polypeptides with 88 % identity and 95 % similarity). ClustalW consensus for the two sequences (Fig 3) delimits a couple of stretches of nonidentity. It should therefore be feasible to decipher the residue(s) that contribute to the origin of substrate cooperativity (Pawlyk & Pettigrew 2002) in Aspergillus NADP-GDH, through site directed mutagenesis.

Acknowledgements We thank Ciba Research (India) Pvt. Ltd., India for help in supply and synthesis of reactive dye coupled matrices, CR-D and CibaFixAR-017. This work was supported under a project and a research fellowship (to R.C.) from CSIR, India.

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