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Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation
Biochimica et Biophysica Acta 1810 (2011) 1346–1354
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation Shinjinee Sengupta, Sagar Lahiri, Shakri Banerjee, Bipasha Bashistha, Anil K. Ghosh ⁎ Biotechnology Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700 032, India
a r t i c l e
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Article history: Received 1 May 2011 Received in revised form 15 June 2011 Accepted 30 June 2011 Available online 13 July 2011 Keywords: Candida utilis Trehalose Trehalose-6-phosphate synthase Arginine
a b s t r a c t Background: Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6phosphate phosphatase (TPP). Methods: In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM L-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). Results: An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37 °C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl2, MgCl2 and ZnSO4, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest Vmax and lowest Km values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. General significance: Substrate specificity, Vmax and Km values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside) is an important disaccharide found widely in nature among yeast, bacteria, fungi, plants and lower animals [1]. It is accumulated during periods of nutrient starvation, desiccation and after exposure to mild heat shock [2]. The sugar serves as a stabilizer of cellular structures under stress conditions besides having an exceptional capacity to protect biological membranes and enzymes from the adverse effects of freezing or drying [3]. It also protects cells from stress induced by exposure to oxygen radicals. [1]. Recently, trehalose is reported to reduce several neural diseases by inducing autophagy including Parkinsonism, Alzheimer's disease etc. [4,5]. The trehalose pathway consists of only a few metabolites, which form a substrate cycle, and is governed
Abbreviations: TPS, Trehalose-6-phophate synthase; TPP, Trehalose phosphate phosphatases; UDPG, Uridine diphosphaphate glucose; ADPG, Adenosine di phosphate synthase; G-6-P, Glucose-6-phophate; F-6-P, Fructose-6-phosphate ⁎ Corresponding author. Tel.: + 91 33 2499 5787/5870; fax: + 91 33 2473 5197. E-mail addresses: [email protected], [email protected] (A.K. Ghosh). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.06.025
by a surprisingly complex control system that comprises of several inhibiting or activating mechanisms [6]. The best known and most widely distributed pathway involves the transfer of glucose from uridine diphosphate glucose (UDPG) to glucose-6-phosphate (G-6-P) to form trehalose-6-phosphate (T-6-P) and uridine diphosphate (UDP) [7]. This step is catalyzed by the enzyme trehalose-6phosphate synthase (TPS). Trehalose-6-phosphate is subsequently dephosphorylated in the next step to yield inorganic phosphate and trehalose. The enzyme trehalose-6-phosphate phosphatase (TPP) catalyzes this conversion. It was reported in yeast that the enzymes involved in trehalose biosynthesis, viz. TPS and TPP exist together in the trehalose synthase complex that is highly regulated at the activity level as well as at the genetic level [8]. The trehalose synthase complex was so far purified as a single unit and the sub units could not be separated from the complex [7,8]. However, TPS was purified in its free form in our laboratory from S. cerevisiae [9]. The present report also deals with TPS purified in its free form from C. utilis. This purification excluded ammonium sulfate fractionation; instead a new methodology of cell lysis as well as HPGFLC in the presence of 100 mM arginine was introduced. L-Arginine is one of the most commonly
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used and most generally applied suppressors of protein aggregation [10]. It is frequently used as a solution additive to stabilize proteins against aggregation. The new purification protocol includes L-arginine so as to avoid the problems faced by protein aggregation phenomenon. The present investigation thus deals with purification, effects of aggregation and dependence on different factors for optimal TPS activity from C. utilis. 2. Materials and methods Trehalose-6-phosphate, uridine diphosphate glucose (UDPG), glucose-6-phosphate (G-6-P), phenylmethylsulphonyl fluoride (PMSF), benzamidine hydrochloride, 2-mercaptoethanol, glycerol, polyethylene glycol, Bradford reagent were obtained from Sigma, USA. Anthrone was procured from Aldrich, USA. Polyacrylamide gel electrophoresis reagents and chemicals were obtained from BIORAD, USA. All the other chemicals and medium components used were of analytical grade and were purchased locally.
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excluding arginine, pH 7.0. Fractions of 0.5 ml were collected and assayed for TPS activity. Active fractions were pooled and concentrated by lyophilization. 2.3.4. Re-chromatography on Biosuite 125 of Step 3 enzyme The concentrated enzyme preparation of Step 3 enzyme was passed again through Biosuite 125 with same flow rate and mobile phase as above. The active enzyme fraction was collected, pooled and stored at −20 °C for future use. 2.3.5. High performance reverse phase liquid chromatography on Delta Pak C4 The Step 4 enzyme was desalted and injected in a HPRPLC column, Deltapak C4 (300 Å, 3.9 × 300 mm, Waters, USA). Two solvents used for HPRPLC were Solvent A (Water containing 0.1% TFA) and Solvent B (Acetonitrile containing 0.1% TFA). Protein was eluted by a 60 min linear gradient to reach from 100% A to 100% B. Flow rate was maintained at 1 ml/min. A214 and A280 were monitored as stated earlier.
2.1. Organism and culture conditions 2.4. Assay of TPS activity The wild type diploid C. utilis (Cat no. NCIM/ Y500) strain used for work was the same as used in previous reports [11]. Cells were grown in YPD medium at 30 °C as stated earlier till desired stage of growth and measured by taking absorbance at 660 nm. Cells were harvested at 10,000 × g at 4–5 °C for 10 min.
TPS activity was assayed at 37 °C for 15 min using 5 mM UDPG and 5 mM G-6-P as substrates [9]. T-6-P formed was determined by anthrone color reagent after neutralizing all other sugars [12–14]. Unit of enzyme activity (U) was expressed in micromole of T-6-P synthesized per minute under assay conditions.
2.2. TPS production during growth 2.5. Protein estimation Growth curve of C. utilis strain was monitored according to the protocol mentioned in the previous reports [9]. Aliquots were collected at different hours of growth and cells were harvested by centrifugation.
Protein was measured by Bradford reagent as per technical bulletin provided by the manufacturer, Sigma. Protein content of whole cell homogenate was determined by the modified method of Lowry [15]. BSA was used as standard.
2.3. Purification of trehalose-6-phosphate synthase 2.6. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis 2.3.1. Preparation of crude extract Cells grown in YPD medium up to A660 ~ 25 were harvested, washed and suspended in ice cold lysis buffer pH 9.6 (20 mM Tris–HCl, buffer containing 1 mM EDTA, 1 mM benzamidine hydrochloride, 1 mM PMSF, 15 mM 2-mercaptoethanol, 10% (w/v) glycerol, 0.1% (w/v) Tween 40 and 100 mM arginine). The cells were lysed by passing twice through a FRENCH Pressure Cell Press (SLM Instruments, USA) at 18,000 psi and observed under microscope to confirm successful lysis. The homogenate was centrifuged at 10,000 × g for 5 min. The supernatant containing nearly 100% activity was designated as crude enzyme solution or Step 1 enzyme. 2.3.2. HPGFLC of Step 1 enzyme The crude enzyme solution from Step 1 was applied to high performance gel filtration liquid chromatography (HPGFLC) using HiLoad 16/60 Superdex 200 preparative grade column (120 ml bed volume) obtained from Amersham Biosciences, Sweden. HPLC buffer, pH 9.6 (Tris–HCl 20 mM, containing 0.1 mM Benzamidine hydrochloride, 0.1 mM PMSF, 0.5 mM 2-mercaptoethanol, 0.5% (w/v) glycerol, 0.1% (w/v) Tween 40 and 100 mM Arginine) was used as mobile phase at a flow rate of 1 ml/min. Eluted fractions were collected and A280 was measured. Fractions containing TPS activity were pooled and concentrated by polyethylene glycol (mol wt ~ 15,000–20,000 Da). 2.3.3. High performance gel filtration liquid chromatography on Biosuite 125 of Step 2 enzyme The concentrated enzyme preparation of Step 2 was passed through another HPGFLC column Biosuite 125 and monitored using dual wavelength detector, both of which have been obtained from Waters, USA, with a flow rate of 0.5 ml/min using same mobile phase but
Non denaturing PAGE of Step-4 TPS was carried out at pH 7.0 on a 7.5% polyacrylamide gel using 8 × 8 × 1.5 mm gel slab Mighty Small gel apparatus obtained from Hoefer. Electrophoresis was carried out at a constant current of 20 mA/slab and according to the guidelines in the technical bulletin provided by the manufacturer (Hoefer, USA). The movement of the proteins entirely depended on the net average negative charge exhibited in the presence of glycine (Tris–glycine buffer, pH 8.5). The single protein band was visualized on staining with Coomassie Brilliant Blue R-250 (Pierce, USA). Denaturizing sodium dodecyl sulfate-polyacryalamide gel electrophoresis (SDS-PAGE) of purified protein sample of Step 4 was carried out at pH 8.5 on a 12.5% resolving gel (8 × 8 × 1.5 mm) preparation using the same Mighty Small electrophoresis unit, Hoefer, USA and discontinuous Laemmli buffer as per GE Healthcare technical bulletin. Denatured protein bands were resolved by a constant supply of current (20 mA/slab) according to the technical bulletin provided by the manufacturer (Hoefer, USA). 2-Mercaptoethanol (5%) and ethylenediaminetetraacetic acid (EDTA; 1 mM) were added to the sample buffer as a solubilizing agent. Low molecular weight markers obtained from GE Healthcare (Code RPN 755), USA, were loaded and run in parallel lanes and gels were stained with Coomassie Brilliant Blue R250 (Pierce, USA) by shaking overnight at 37 °C. 2.7. Molecular weight estimation Molecular weight of the purified enzyme protein was estimated by HPGFLC from a plot of log of molecular weight versus Kav values of low molecular weight standards (GE Healthcare, USA). Molecular weight
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was also estimated by SDS-PAGE from a plot of Rf (relative migration values) versus log of molecular weight of LMW gel electrophoresis markers [GE Healthcare, USA]. 2.8. N-terminal sequencing The purified protein eluting out from HPRPLC was spotted on a PVDF membrane following the method suggested by the manufacturer [16]. The membrane was washed with double distilled water as per the method suggested by the manufacturer. The transferred protein bands on PVDF were stained and identified with Ponceau S solution [16]. The identified protein bands of interest were excised for N-terminal sequencing studies. The desired bands were analyzed by N-terminal sequencing using a Model 491 Procise protein/peptide sequencer from Applied Biosystems, USA. The sequence obtained from the N-terminal sequencer was matched with the database of NCBI (National Center for Biological Information) using BLAST (Basic Local Alignment Search Tool). BLAST was also used to find the similarity of the N-terminal sequence obtained here with the protein sequence of known TPS from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe. 2.9. Physicochemical characterization 2.9.1. Determination of temperature and pH optima Optimum pH was determined by measuring TPS activity at 37 °C for 15 min at different pH ranging from 5.0 to 9.5 using the following buffer systems: 0.05 M sodium acetate buffer (pH 5.0 to 5.5), 0.05 M sodium phosphate buffer (pH 6.0 to 6.5) and 0.05 M Tris–HCl buffer (pH 7.0 to 9.5). The pH corresponding to maximum activity was taken as the optimum pH. Optimum temperature was determined by measuring TPS activity at various temperatures (10 °C to 80 °C) for 15 min at pH 8.5. The temperature corresponding to maximum activity was taken as the optimum temperature. 2.9.2. Determination of temperature and pH stability For temperature stability, enzyme solution taken in 50 mM Tris–HCl buffer (pH 8.5) was pre-incubated at different temperatures (0 °C to 60 °C) for 15 min. Samples were then maintained at a temperature of 37 °C and TPS assay was performed by the addition of substrates and co-factors. TPS activities of these pre-incubated samples were measured. The stability of TPS activity was studied by keeping the enzyme solution for 30 min at 37 °C in different pH solutions ranging from pH 4.5 to 10.5. Buffers used were as follows: 0.05 M sodium acetate buffer (pH 4.5 to 5.5), 0.05 M sodium phosphate buffer (pH 6.0 to 7.0), 0.05 M Tris–HCl buffer (pH 7.0 to 9.5) and 0.05 M glycine–NaOH buffer (pH 10.5). The samples were assayed normally by adding substrates (UDPG and G-6-P) and co-factors (MnCl2 and heparin) as described earlier. TPS activities of these pre-incubated samples were measured. 2.9.3. Substrate specificity of enzyme Sugar nucleotides UDPG, ADPG, GDPG or TDPG were used as glucosyl donors and enzyme assays were carried out as described previously. In all sets, G-6-P was the universal glucosyl acceptor. In another set of experiments, UDPG was kept as the universal glucosyl donor and G-6-P, fructose-6-phosphate, (F-6-P), mannose-6-phosphate (M-6-P) or glucosamine-6-phosphate were used as glucosyl acceptors. TPS activity was represented as U/mg. 2.9.4. Requirement of polyanions for TPS activity Effect of polyanions on different glucosyl donors of TPS was determined. Assay mixtures contained 0 to 1 μg of either heparin or chondroitin sulfate or RNA along with 5 mM of G-6-P and various sugar nucleotides. Other assay components were not altered. Tris–HCl
buffer 50 mM, pH 8.5 was used as the assay buffer. Values were represented as U/mg. 2.9.5. Requirement of metal co-factors for TPS activity Activity of purified TPS was assayed at 37 °C for 15 min using 5 mM UDPG and G-6-P as substrates along with different metal salts. Assay mixture without any added metal salts was considered as the control set. Values were represented as U/mg. 2.9.6. Effect of metal chelators and inhibitors on TPS activity Purified TPS enzyme was pre-incubated with specific concentrations of different compounds in 50 mM Tris–HCl buffer (pH 8.5) for 15 min at 37 °C unless otherwise stated. Assay was started with the addition of the substrate and co-factors. Residual activities of the preincubated samples were measured. Values were expressed in percentage and TPS activity of a sample identically pre-incubated but in the absence of any compound was considered as 100%. 2.9.7. Enzyme progress curve of TPS TPS activity was measured using 0.94 μg of enzyme taken in a 100 μl of assay mixture and assayed as above. Assay mixtures were incubated for different time intervals as indicated in Fig. 7. TPS activity values were expressed as U/mg. 2.10. Regulation of trehalose-6-phosphate synthase 2.10.1. Effect of thiol modifiers on purified TPS activity Purified TPS was pre-incubated with 1 mM concentration of various thiol modifiers in 50 mM Tris–HCl buffer pH 8.5 for 15 min at 37 °C. Assay was started with the addition of the substrates and cofactors. Residual activities of the pre-incubated samples were measured. Values were expressed in percentage and TPS activity of a sample identically pre-incubated but in the absence of any chemical was considered as control. 2.10.2. TPS activity as a function of protein concentration TPS activity was measured using different concentrations of protein in 100 μl of assay mixture. Assay was done as stated above. TPS activity values were expressed as U/mg. The data obtained from the experiment of temperature stability was plotted in an Arrhenius plot and energy of activation (Ea) was calculated from the slope of the plot following a previously reported protocol [17]. 3. Results 3.1. TPS product during growth Rate of cell division increased rapidly during the exponential phase and then gradually slowed down till it plateaued during the stationary phase. TPS activity in cell lysate started increasing 10 h onwards and peaked at 36 h after inoculation when A660 values of the culture corresponded to ~25 after which it was seen to fall gradually (Fig. 1). Maximum specific activity of TPS was 0.096 U/mg protein at A660 ~ 25. 3.2. Purification of trehalose-6-phosphate synthase 3.2.1. HPGFLC of Step 1 enzyme Step 1 enzyme (cell lysate) was subjected directly to high performance gel filtration liquid chromatography (HPGFLC) using HiLoad 16/60 Superdex 200 preparative grade column from Amersham Biosciences, Sweden at a flow rate of 1 ml/min. Three protein peaks were obtained at 40, 67 and 113 min of elution (data not shown). TPS activity eluted out in the second peak (64–70 ml). The other two peaks contained no TPS activity. This was designated as step 2.
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activity 2.03 U/mg with a 22.1 purification fold and yield of 53.11% (Table 1).
3.2.5. High performance reverse phase liquid chromatography on Delta Pak C4 The Step 4 enzyme was subjected to reverse phase chromatography in a Delta Pak C4 column (Waters, USA). A sharp peak was obtained at 18.5 min (Fig. 2). The 36 min and 45 min peaks seen were obtained in blank run as well. Fig. 1. Growth curve of C. utilis stain. Growth was monitored by measuring absorbance at 660 nm (—♦—). Specific activity of TPS (—■—) was measured at different phases of growth. All experiments were conducted in triplicate.
3.2.2. High performance gel filtration liquid chromatography (HPGFLC) on Biosuite 125 of Step 2 enzyme The Step-2 TPS obtained above was concentrated by PEG and a second HPGFLC was performed with the concentrated Step-2 enzyme to analyze the effect of a second gel filtration on enzyme activity. Chromatographic profile showed a minor peak at filtration at 12.2 min and a sharp major peak at 13.8 min (data not shown). TPS activity was found to be eluted out at the second major peak from 13 to 16 min. This purification step was designated the Step-3 TPS. 3.2.3. Re-chromatography on Biosuite 125 of Step 3 enzyme The Step-3 TPS obtained above was again concentrated by PEG, and a third HPGFLC was performed to obtain a single peak with TPS activity at 13.6 min (Fig. 2). This finally purified TPS is designated as the Step 4 enzyme. 3.2.4. Purification table of TPS The purification table enumerates the four steps of the purification protocol and mentions total protein, total enzyme activity, specific activity as U/mg, purification fold and yield obtained at each purification step. Final purification step (step 4) contained specific
3.3. Native and sodium dodecyl sulfate-polyacryalamide gel electrophoresis Non denaturing gel electrophoresis of Step 4 enzyme showed a single band confirming the purity of the enzyme (Fig. 3A). SDS PAGE analysis of Step 4 enzyme showed a single band and which was calculated as 60 kDa when compared with standard protein markers (LMW gel electrophoresis markers) thereby confirming the purity of the enzyme (Fig. 3B).
3.4. N-terminal sequencing A 20 residue long amino acid sequence of the N-terminal of the protein was obtained as ‘DTVESEIAFVFDDLGEEXFF’. BLAST analysis with NCBI database revealed that none of the non redundant protein sequences matched completely with the sequence obtained from the N-terminal sequencing which was expected since no TPS sequence from C. utilis is available yet. BLAST analysis was also done to match the sequence with known protein sequences of TPS from related species of yeast which revealed that the experimentally obtained N-terminus of the purified protein was most similar to TPS from S. cerevisiae followed by TPS from S. pombe (Table 2). The alignment of the N-terminal sequence with these two sequences was also matched using EMBOSS Pairwise Alignment Algorithm Needle.
Fig. 2. Step 5: Re-chromatography on Biosuit 125 and high performance reverse phase liquid chromatography on Delta Pak C4. Enzyme solution obtained from step 3 was separated by using HPGFLC column Biosuit 125 as described in the text. Enzyme solution obtained from step 4 was separated by using HPRFLC column Delta Pak C4 as described in the text.
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Table 1 Purification table of TPS from C. utilis. Steps
Total protein (mg)
Total enzyme activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
Soluble supernatant (Step 1) HPGFLC on Hiload 16/60 Superdex 200 (Step 2) HPGFLC Biosuite 125 (Step 3) HPGFLC Biosuite 125 (Step 4)
183.2 ± 0.81 12.1 ± 0.76 4.1 ± 0.61 2.9 ± 0.33
17.6 ± 0.31 13.9 ± 0. 26 7.4 ± 0.22 5.9 ± 0.17
0.096 ± 0.001 1.14 ± 0.002 1.804 ± 0.09 2.03 ± 0.07
1 11.97 18.84 22.192
100 74.25 64.22 53.11
Purification was performed as described in the text. Values represented are average of three sets of purification data. The standard deviation of each measurement is indicated.
3.5. Determination of temperature and pH optima The pH optimum was recorded at pH 8.5 with 1.97 U/mg activity (data not shown). TPS activity was seen to be more affected at low pH conditions. The Step-4 TPS enzyme displayed a classical bell curve of temperature optima. Highest TPS activity was observed at 37 °C with 1.99 U/mg activity. Very low activities of 0.4 U/mg were detected at 10 °C and 60 °C beyond which no activity of TPS was seen (data not shown). 3.6. Determination of temperature and pH stability The Step 4 enzyme activity was more or less stable in the temperature range of 0–40 °C (data not shown). It showed a gradual decrease in stability with increasing temperature. Even at 60 °C, around 40% activity was retained. TPS activity appeared to be maximally stable at pH 8.5 with 2.11 U/ mg activity, followed by 2.01 U/mg activity at pH 9.0 and 1.93 U/mg activity at pH 9.5 (data not shown). In the highly alkaline condition of pH 10.5, 1.21 U/mg activity was observed. Stability of TPS activity was more affected at lower pH conditions. Only 0.31 U/mg activity was retained at pH 4.5. 3.7. Substrate specificity of TPS TPS activity was assayed using different sugar nucleotides to observe whether they could act as potential glucosyl donors for the purified enzyme. Maximum TPS activity was obtained with ADPG (~3.5 U/mg), while with UDPG, the activity was nearly halved to 1.9 U/mg (Fig. 4A). GDPG gave 0.9 U/mg activity while TDPG showed the least effect on TPS by displaying the lowest activity (0.6 U/mg) among the four. Substrate specificity of TPS with respect to its glucosyl
acceptor displayed a specific activity of ~ 2.6 U/mg with G-6-P while F-6-P displayed an activity of ~ 1 U/mg which was ~ 2.5 times less the activity obtained with G-6-P (Fig. 4B). M-6-P and Glucosamine-6-P were not glucosyl acceptors for TPS activity. The initial velocity (v) of the enzyme activity was seen to increase gradually with increasing concentration of UDPG and G-6-P from 0.5 mM to 20 mM. Km value for ADPG was determined as 1.36, while those for UDPG, GDPG and TDPG were observed to be 2.15, 2.24 and 2.81 respectively (Table 3). Lineweaver Burk's plots displayed Vmax of purified TPS for different glucosyl donors such as 2.96 for ADPG, 2.41 for UDPG, 2.37 for GDPG and 2.35 and TDPG (Table 3). Km and Vmax values were different from those obtained from TPS from S. cerevisiae [18]. Km values for S. cerevisiae TPS is 2 mM for UDPG since ADPG is not the substrate [18]. 3.8. Requirements of metal co-factors for TPS activity Assay mixtures contained 5 mM of respective metal salts as mentioned in Table 4. Assay mixture without any added metal was considered as the control set. TPS activity was markedly stimulated with 5 mM MnCl2, and showed maximum activity of 1.513 U/mg. The enzyme was relatively specific for MnCl2, and was much less active in case of other metals. Most metals used were not able to replace MnCl2, or were only slightly effective in this regard. MgCl2, at 5 mM concentration, was also effective and displayed TPS activity of 1.47 U/mg. Optimum concentration in case of both MnCl2 and MgCl2 was seen to be 70 mM (Fig. 5). TPS activity in the presence of ZnSO4 and CuSO4 displayed greater activity than that of control. Several metals were seen to display lower activity than that of control, suggesting a potential inhibition of enzyme activity. These metals included CdCl2 and AgNO3 being the most potent. 3.9. Dependence on polyanion concentrations for optimum TPS activity Heparin, chondroitin sulfate and RNA were used as polyanions. Assay mixtures contained heparin from 0 to 1 μg along with 5 mM of G-6-P and different sugar nucleotides UDPG, GDPG, ADPG and TDPG. As observed previously, ADPG displayed maximum TPS activity with heparin. ADPG was observed to be the best substrate, and displayed maximum activity of 4.23 U/mg at 0.15 μg heparin, followed by UDPG (2.016 U/mg) and GDPG (1.609 U/mg). TDPG displayed maximum activity of 0.95 U/mg at 2 μg (Fig. 6A). TPS activity fell after 0.20 μg heparin concentration. Similar results were observed with
Table 2 Summary of the BLAST analysis.
Fig. 3. Gel electrophoresis: SDS and native. Non denaturing PAGE and SDS PAGE were carried out as described in the text. SDS PAGE of step 4 enzyme and low molecular weight markers were run. Protein standards were from the LMW calibration kit from Amersham Biosciences, UK. Corresponding molecular weights are indicated next to the protein bands. Marker lanes in SDS PAGE were considered for molecular weight estimation.
Accession no.
Organism
Max score
Total score
Query coverage (%)
E value
ADM26405
Saccharomyces cerevisiae Schizosaccharomyces pombe Candida albicans
16.3
41.4
28
0.14
11.2
19.5
53
4.8
10.8
31.8
35
6
P40387 Q92410
N-terminal sequence obtained was matched with the database of NCBI. Sequences were matched using BLAST (Basic Local Alignment Search Tool). Details of the matches, TPS from S. cerevisiae, C. albicans and S. pombe are given.
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Table 4 Effects of cofactors on TPS activity. Metal
Concentration (mM)
Specific activity (U/mg)
Activity (%)
None MnCl2 MgCl2 ZnSO4 CuSO4 CoCl2 CaCl2 BaCl2 FeSO4 CdCl2 AgNO3
– 5 5 5 5 5 5 5 5 5 5
0.589 ± 0.2 1.513 ± 0.22 1.47 ± 0.17 1.314 ± 0.19 0.786 ± 0.32 0.641 ± 0.35 0.631 ± 0.3 0.567 ± 0.16 0.524 ± 0.21 0.461 ± 0.22 0.32 ± 0.23
100 256.87 249.57 223.1 133.45 108.83 107.13 96.265 88.96 78.27 54.33
TPS assay was done as described in the text. Sets containing purified TPS were preincubated for 15 min at 37 °C. All experiments were performed in triplicate. Standard deviation of each measurement is indicated.
3.12. Regulation of trehalose-6-phosphate synthase
Fig. 4. Substrate specificity of TPS. Enzyme assays were performed as described in the text. (A) Sugar nucleotides ADPG, UDPG, GDPG and TDPG were used as glucosyl donor. (B) Sugar phosphates G-6-P, F-6-P and M-6-P were used as glucosyl acceptor. In both cases, white bars denote enzyme activity in U/mg. Data given are the average of three sets of values, which did not vary more than ±5% represented by error bars.
chondroitin sulfate as well (Fig. 6B). An increase in TPS activity was seen up to 0.2 μg post which fell with increasing concentration. ADPG displayed the highest activity of 3.8 U/mg followed by UDPG (2.8 U/ mg). GDPG and TDPG displayed activity values of 2.1 U/mg and 1.1 U/ mg respectively. RNA had no effect on the activity of TPS.
3.12.1. Effect of thiol modifiers on TPS activity TPS activity was seen to enhance in the presence of IAA and IAM (Table 6). When 1 mM concentration of IAA was used, TPS was observed at 147% residual activity. IAM also enhanced TPS activity displaying 139% of residual activity. However, when 1 mM NEM was used, TPS activity was inhibited resulting in only 32.41% of residual activity. 3.12.2. TPS activity as a function of protein concentration TPS activity was measured using different concentrations of protein in 100 μl of assay mixture. Activity could only be detected when protein concentration was increased to 0.2 μg of protein. No activity was detected when protein concentration was less than 0.2 μg. When 0.50 μg protein was used, increase in specific activity was seen. Further increasing protein concentration to 1 μg protein resulted in the highest specific activity of TPS. However, specific
3.10. Effect of metal chelators and inhibitors on TPS activity Purified TPS was pre-incubated with different metal chelators and inhibitors (Table 5). Some of the metal chelators were seen to increase TPS activity over control like EGTA and CGTA with residual activity as 129% and 141% respectively. Citric acid and EDTA were seen to inhibit TPS activity as they were observed to display 79% and 80% residual activity.
3.11. Enzyme progress curve of TPS An enzyme progress curve was prepared using different incubation time. TPS activity was seen to maintain linearity from 3 min to 15 min of incubation time after which the linearity was lost (Fig. 7). Table 3 Effect of substrate concentration on reaction velocity. Glucosyl donors
Vmax (U/mg)
Km (mM)
ADPG UDPG GDPG TDPG
2.96 ± 0.07 2.41 ± 0.09 2.37 ± 0.01 2.35 ± 0.08
1.36 ± 0.08 2.15 ± 0.01 2.24 ± 0.02 2.81 ± 0.08
Michaelis constant (Km) and maximum velocity (Vmax) for TPS activity measured at increasing concentration of ADPG, UDPG, GDPG and TDPG keeping G-6-P concentration constant at 5 mM. Data given are average of three sets of values. The standard deviation of each measurement is indicated.
Fig. 5. Effect of MgCl2 and MnCl2 on TPS activity. (A) Effect of MgCl2 on TPS activity was studied using 0, 10, 20, 50, 75 and 100 mM of the salt. (B) Effect of MnCl2 on TPS activity was studied using 0, 10, 20, 50, 75 and 100 mM of the salt. Assays were performed as described in the text. Data given are the average of three sets of values, which did not vary more than ± 5% represented by error bars.
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Fig. 7. Enzyme progress curve of TPS. Enzyme assays were performed as described in the text but with different incubation times. Assay mixture reactions were stopped at different time intervals of 0, 3, 5, 10, 15, 20, 30, 35 and 60 min. Data given are the average of the three sets of values, which did not vary more than ±5% represented by error bars.
Fig. 6. Effect of polyanions on different glucosyl donors. Enzyme assays were performed as described in the text. (A) Heparin at different concentrations of 0, 0.05. 0.1, 0.15, 0.2, 0.5 and 1 μg were taken. Glucosyl donors used were ADPG (—♦—), UDPG (—■—), GDPG (—●—) and TDPG (—▲—). (B) Chondroitin sulfate at different concentrations of 0, 0.05. 0.1, 0.15, 0.2, 0.5 and 1 μg were taken. Glucosyl donors used were the same as for heparin. Data given are the average of three sets of values, which did not vary more than ± 5% represented by error bars.
activity decreased beyond this point on further increasing protein concentration (data not shown). An Arrhenius plot of this enzyme showed a sharp discontinuity after 40 °C. Activation energy at 5 to 40 °C was calculated to be 35.65 cal/mol. At temperature 50 to 60 °C, activation energy was calculated as 7885.6 cal/mol (Fig. 8). 4. Discussion Trehalose is a multifunctional sugar which is biosynthesized in S. cerevisiae during the beginning of the stationary phase [9,19]. Since the synthesis of sugar is catalyzed by the enzymes trehalose-6phosphate synthase (TPS) and trehalose-6-phoshate phosphatases (TPP), the level of trehalose in the cell is dependent on the activity profiles of these enzymes. The present study reports purification and characterization of only TPS in its free form from C. utilis. Growth curve of C. utilis showed that the TPS activity was highest at A660 ~ 25 which was obtained at 36 h of growth, after which it was seen to fall gradually (Fig. 1). Thus, all the purification experiments of TPS were initiated at A660 ~ 25. TPS purified from S. cerevisiae attained the highest level at 33 h when A660 ~ 18 [9].
Table 5 Effect of chelators and inhibitors on TPS activity. Serial Chemical added
Conc. (mM) Activity (U/mg) Residual activity (%)
1. 2. 3. 4. 5. 6. 7. 8.
– 2.5 2.5 2.5 2.5 2.5 2.5 2.5
None CDTA EGTA 5′-sulfosalicylic acid Tri sodium citrate Sodium azide EDTA Citric acid
1.45 ± 0.11 2.05 ± 0. 19 1.88 ± 0.14 1.527 ± 0.13 1.423 ± 0.23 1.17 ± 0.22 1.16 ± 0.14 1.15 ± 0.19
100 141.25 129.56 105.31 98.12 81.21 80.15 79.14
TPS assay was done as described in the text. Sets containing TPS were pre-incubated for 15 min at 37 °C. All experiments were performed in triplicate. The standard deviation of each measurement is indicated.
Many reports of TPS purification from various organisms are reported to date. Londesborough and Vuorio obtained a 3 fold purification of TPS from S. cerevisiae [7]. TPS was also purified by Vandercammen et al. with 40 fold purification from S. cerevisiae [20]. 105 fold purified TPS has been reported from a basidiomycetous fungus Grifola frondosa [21]. Earlier reports from our laboratory reported the isolation of TPS enzyme from the trehalose synthase complex in its free form from S. cerevisiae for the first time [9]. Here, we have reported the purification of TPS from C. utilis for the first time by using a new purification strategy. A high purification fold of 22.1 was achieved as compared to 15.8 purification fold reported previously from our laboratory (Table 1) [9]. This makes this purification strategy better than the previous one reported from our laboratory. The purification process excluded ammonium sulfate fractionation, and instead introduced a new methodology of cell lysis as well as HPGFLC in the presence of 100 mM arginine. When purification of TPS was performed with the traditional protocol it resulted in a protein peak at 12.1 min during final HPGFLC which showed reduced enzyme activity and subsequent degradation with time (data not shown). This observation might be due to proteolysis or a protein aggregation phenomenon. Thus, to avoid this consequence, arginine was introduced in cell lysis as well as HPGFLC buffer. Arginine is known to have an anti-aggregation property and its role as an aggregation suppressor has been reported in detail [10]. Hence, the presence of 100 mM arginine in the initial purification step resulted in a shift of the protein peak to 13.6 min with stable activity during final HPGFLC (Fig. 2). This also resulted in improved yield due to suppressed aggregation as evident from the protein peak shift. A single band was visualized in both native and SDS PAGE confirming the purity of the Step 4 enzyme. The molecular weight of the enzyme was calculated as 60 kDa (Figs. 2 and 3). To confirm the identity of the purified protein as TPS, N-terminal sequence analysis of the 60 kDa band was performed. A 20 residue long sequence was obtained from the N terminus and this displayed various levels of similarity with different known sequences of TPS obtained from different yeasts. Around 53% of query coverage and 4.8 E value were obtained with S. pombe (Table 2). 35% query coverage
Table 6 Effect of thiol modifiers on purified TPS activity. Thiol modifiers
Conc (mM)
Activity (U/mg)
Residual activity (%)
None (control) Iodoacetic acid Iodoacetamide N-ethylmaleimide
0 1.0 1.0 1.0
1.45 ± 0.3 2.14 ± 0.32 2.02 ± 0.27 0.47 ± 0.21
100 147.5 139.3 32.41
TPS assay was done as described in the text. Sets containing TPS were pre-incubated for 15 min at 37 °C. All experiments were performed in triplicate. The standard deviation of each measurement is indicated.
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Fig. 8. TPS activity as a function of temperature (Arrhenius plot). Activity was measured as stated in the Materials and methods with different temperatures (5–60 °C). Values were obtained from the average of three sets of triplicates. The activation energy (Ea) values (cal/mol) were calculated from the slope of the plots and are shown in the inset.
and 6 E-value were obtained with C. albicans and 28% query coverage with S. cerevisiae and lowest E-value of 0.14 showed its maximum resemblance with the purified TPS from S. cerevisiae. Since comparison of 20 amino acid residues showed more resemblance with the TPS sequence from S. cerevisiae, it was supposed that the 60 kDa band which had been purified and isolated from the complex was indeed TPS. A different physicochemical characterization of the purified TPS was performed to obtain a better idea of the enzyme. The pH optimum of TPS from C. utilis was similar to that of TPS from S. cerevisiae and TPS from M. smegmatis (data not shown) [18,12]. The temperature optimum of TPS from C. utilis showed a similarity with that of TPS from S. cerevisiae [18] and basidiomycetes fungus G. frondosa [21] (data not shown). The C. utilis TPS was stable at 40 °C for 15 min, in comparison to G. frondosa TPS, where nearly all activity was lost after 30 min incubation at 40 °C [21]. This observation was similar to TPS from S. cerevisiae [18]. Purified C. utilis TPS was also observed to be analogous to S. cerevisiae and G. frondosa TPS in terms of their pH stability, as both demonstrated more stability at alkaline pH [18,21]. After the identification of the purified enzyme as TPS, different possible substrates of the enzyme were identified other than the ubiquitous UDPG and G-6-P. Substrate specificity experiments were performed both for glucosyl donors as well as acceptors, and varying levels of activity with different substrates were obtained. This study showed a remarkable observation where TPS activity was at the maximum with ADPG than its usual substrate UDPG (Fig. 4). The same enzyme also showed activity with GDPG and TDPG. TPS with ADPG as a substrate showed high Vmax and a low Km value confirming it to be the ideal substrate for this enzyme (Table 3). TPS from S. cerevisiae also showed activity with different glucosyl donors except ADPG, which was not a substrate [18]. Thus, these observations provided an insight into an altered trehalose pathway occurring in C. utilis using ADPG as a substrate instead of UDPG. Similar observations are reported in some mutant strains of S. cerevisiae where ADPG dependent trehalose synthesis pathway is present [22]. Since in these mutants the normal trehalose synthesis pathway is disrupted, trehalose synthesis via ADPG dependent trehalose synthase was preferred [22]. In the present study, pure TPS was able to carry out independent conversion of F-6-P to G6-P. It was known that TPS had some associated isomerase activity and is reported to be a bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI) from S. cerevisiae [18,23]. Metal ions function as enzyme co-factors either by binding to the actual catalytic site of the enzyme or by converting the enzyme into its active form without being directly involved in catalysis [24]. Previous reports showed that the requirement for metal co-factors was not mandatory but the presence of some metals like MnCl2 and ZnCl2 enhanced TPS activity in S. cerevisiae [18]. In the present report also, the effects of co-factors has been established as not mandatory for TPS activity (Table 4). However, the presence of some metals such as
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MnCl2 and MgCl2 enhanced TPS activity. There are reports of MnCl2 as a co-factor of TPS activity from M. smegmatis [12]. Metals like CdCl2, FeSO4, AgNO3 and BaCl2 were possible inhibitors to enzyme activity, as they displayed lower activity than control. Though MnCl2 and MgCl2 were seen to enhance TPS activity, it was, however, observed that both of them caused inhibition of enzyme activity at a high concentration (100 mM) (Fig. 5). A high MnCl2 concentration possibly caused charge re-distribution in the enzyme molecule, resulting in separation of enzyme subunits and decrease in activity [25]. There are several reports of heparin and other associated polyanions affecting TPS activity in mycobacteria as well as S. cerevisiae [14,18]. In the present report the effect of heparin was most prominent with ADPG. A similar pattern was also observed in the case of UDPG followed by GDPG and then TDPG (Fig. 6A). Similar results were obtained when chondroitin sulfate was used instead of heparin (Fig. 6B). These observations are not analogous to the pattern followed in S. cerevisiae where effects of the polyanions were more predominant on UDPG [18]. Activity of TPS was increased in comparison with control in the presence of metal chelators like EGTA and CDTA. The possible explanation behind such an observation might be that these two are specific chelators of those metals which may be potential inhibitors of TPS (Table 5). In contrast, TPS activity deceased in the presence of non specific metal chelators like EDTA, sodium azide and citric acid. Thus, non specific chelation of potential stimulators for TPS possibly resulted in reduced activity upon incubation. These observations were in accordance to S. cerevisiae TPS [18]. Enhancement of enzyme activity by IAA and IAM posed to be an interesting study (Table 6). IAA acts by incorporating an acetic acid group in cysteine and causes carboxymethylation, while IAM incorporates an acetamide group in the amino acid to cause carboxyamidomethylation [26]. NEM modifies the thiol group of cysteine by incorporating a bulky group that does not cause any carboxymethylation [26]. Contrary to IAA and IAM, NEM was seen to inhibit TPS activity. Hence, it appeared that effects of IAA and IAM on TPS were carboxymethylation related. This observation was similar with TPS from S. cerevisiae where TPS activity was enhanced due to carboxymethylation of cysteine residues [18,27]. When protein concentration was increased up to 1 μg TPS activity was seen to increase. However specific activity decreased when protein concentration was further increased. At 2.5 μg protein concentration a ~ 30% decrease in the TPS activity was observed. This showed that increasing the concentration of reacting molecules caused an increase in specific activity up to a certain concentration. It thus appeared that aggregation of protein was important for regulation of TPS activity. TPS showed linearity up to 15 min of incubation time after which linearity was lost (Fig. 7). When TPS was subjected to different temperature changes a sharp discontinuity was observed in the Arrhenius plot corresponding to a large change in the activation energy (Fig. 8). A sudden drop in the Arrhenius plot indicated enzyme deactivation which is due to protein aggregation phenomenon [28]. When an enzyme exhibits an aggregation–de-aggregation state having a different activation energy, then a sharp discontinuity is observed. Since activation energy at 40–60 °C is much higher than that at 5–40 °C, it is expected that at a higher temperature change in enthalpy (ΔH) or entropy (ΔS) will be higher. Hence, the presence of de-aggregated form is expected at a higher temperature and aggregation is favored at a lower temperature. This kind of an identical study is also reported in xylanolytic amyloglucosidase [17]. 5. Conclusion It can be concluded that a new purification strategy is introduced during purification of TPS from C. utilis. This purified TPS exhibited an altered trehalose metabolic pathway using ADPG and G-6-P as
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substrate which forms T-6-P and ADP in the presence of trehalose-6phosphate synthase (TPS). Acknowledgements The authors would like to thank The Director, IICB for financial help from CSIR network project NWP 0005. We are also grateful to Dr. R. Majhi, T.O. IICB for the help provided in N-terminal sequencing. Fellowship provided by CSIR is gratefully acknowledged. References [1] N. Benaroudj, D.N. Lee, A.L. Goldberg, Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals, J. Biol. Chem. 276 (2001) 24261–24267. [2] C. Devirgilo, T. Hottinger, J. Dominguez, T. Boller, A. Wiemken, The role of trehalose synthesis for the acquisition of thermotolerance in yeast, Eur. J. Biochem. 219 (1994) 179–186. [3] E.O. Voit, Biochemical and genomic regulation of the trehalose cycle in yeast: review of observations and canonical model analysis, J. Theor. Biol. 223 (2003) 55–78. [4] J. Rodríguez-Navarro, L. Rodríguez, M. Casarejos, R. Solano, A. Gómez, J. Perucho, A. Cuervo, J. García de Yébenes, M. Mena, Trehalose ameliorates dopaminergic and tau pathology in parkin deleted/tau overexpressing mice through autophagy activation, Neurobiol. Dis. 39 (3) (2010) 423–438. [5] Y. Aguiby, A. Heiseke, S. Gilch, C. Riemer, M. Baier, H.M. Schätzl, A. Ertmer, Autophagy induction by trehalose counteracts cellular prion infection, Autophagy 5 (3) (2009) 361–369. [6] W. Bell, W. Sun, S. Hohmann, Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex, J. Biol. Chem. 273 (1998) 33311–33319. [7] L. Londesborough, O. Vuorio, Trehalose-6-phosphate synthase/phosphatase complex from baker's yeast, J. Gen. Microbiol. 137 (1991) 323–330. [8] E. Cabib, L.F. Leloir, The biosynthesis of trehalose phosphate, J. Biol. Chem. 231 (1958) 259–275. [9] P. Chaudhuri, A. Basu, A.K. Ghosh, Aggregation dependent enhancement of trehalose-6-phosphate synthase activity in Saccharomyces cerevisiae, Biochim. Biophys. Acta 1780 (2008) 289–297. [10] R. Ghosh, S. Sharma, K. Chattopadhyay, Effect of arginine on protein aggregation studied by fluorescence correlation spectroscopy and other biophysical methods, Biochemistry 48 (2009) 1135–1143. [11] A. Roy, S. Bhattacharyya, S. Sengupta, A.K. Ghosh, Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis, World J. Microbiol. & Biotechnol. 17 (2001) 727–729.
[12] D. Lapp, B.W. Patterson, A.D. Elbein, Properties of a trehalose phosphate synthetase from Mycobacterium smegmatis, J. Biol. Chem. 246 (1971) 4567–4579. [13] J.E. Hodge, B.T. Hofreiter, Determination of reducing sugars and carbohydrates, in: R.L. Whistler, M.L. Wolfrom (Eds.), Methods Carbohydr Chem, 1, Acad. Press, New York, 1974, pp. 1380–1390. [14] Y.T. Pan, R.R. Drake, A.D. Elbein, Trehalose-p-synthase of mycobacteria: its substrate specificity is affected by polyanions, Glycobiology 6 (1996) 453–461. [15] H.U. Bergmeyer, E. Bernt, D-Glucose determination with oxidase and peroxidase, in: H.U. Bergmeyer, K. Gaweh (Eds.), 2nd ed, Methods of Enzymatic Analysis, vol. 3, Acad. Press, New York, 1974, pp. 1205–1215. [16] D.W. Speicher, Microsequencing with PVDF membranes: efficient electroblotting, direct protein adsorption and sequencer program modifications, in: T. Hugli (Ed.), Techniques in Protein Chemistry, Academic Press, San Diego, 1989, pp. 24–35. [17] A.K. Ghosh, A.K. Naskar, S. Sengupta, Characterisation of a xylanolytic amyloglucosidase of Termitomyces clypeatus, Biochim. Biophys. Acta 1339 (1997) 289–296. [18] P. Chaudhuri, A. Basu, S. Sengupta, S. Lahiri, T. Dutta, A.K. Ghosh, Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae, Biochim. Biophys. Acta 1790 (2009) 368–374. [19] M. Werner-Washburne, E. Braun, G.C. Johnston, R.A. Singer, Stationary phase in the yeast Saccharomyces cerevisiae, Microbiol. Rev. 57 (1993) 383–401. [20] A. Vandercammen, J. Francois, H.G. Hers, Characterization of trehalose-6phosphate synthase and trehalose-6-phosphate phosphatase in Saccharomyces cerevisiae, Eur. J. Biochem. 182 (1989) 613–620. [21] K. Saito, T. Kase, E. Takahashi, S. Horinouchi, Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa, Appl. Environ. Microbiol. 64 (1998) 4340–4345. [22] J.C. Ferreira, J.M. Thevelein, S. Hohmann, V.M.F. Paschaolin, L.C. Trugo, A.D. Panek, Trehalose accumulation in mutants of Saccharomyces cerevisiae deleted in the UDPG-dependent trehalose synthase–phosphate complex, Biochim. Biophys. Acta 1335 (1–2) (1997) 40–50. [23] J. Londesborough, O. Vuorio, Purification of trehalose synthase from baker's yeast: its temperature-dependent activation by fructose-6-phosphate and inhibition by phosphate, Eur. J. Biochem. 216 (1993) 841–848. [24] H. Suzuki, M.J. Pabst, R.B. Johnston, Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes, J. Biol. Chem. 260 (1985) 3635–3639. [25] R. Swislockaa, E. Oleksinkia, E. Regulskaa, M. Kalinowskaa, W. Lewandowski, Structural characterization of alkali metal 3-nitrobenzoates, J. Mol. Struct. 834–836 (2007) 380–388. [26] J. Bardwell, Thiol modifications in a snapshot, Nat. Biotechnol. 23 (2005) 42–43. [27] S. Sengupta, P. Chaudhuri, S. Lahiri, T. Dutta, S. Banerjee, R. Majhi, A.K. Ghosh, Possible regulation of trehalose metabolism by methylation in Saccharomyces cerevisiae, J. Cell. Physiol. 226 (2010) 158–164. [28] I.H. Segal, Biochemical Calculations, John Wiley and Sons, New York, 1975 278.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation Shinjinee Sengupta, Sagar Lahiri, Shakri Banerjee, Bipasha Bashistha, Anil K. Ghosh ⁎ Biotechnology Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700 032, India
a r t i c l e
i n f o
Article history: Received 1 May 2011 Received in revised form 15 June 2011 Accepted 30 June 2011 Available online 13 July 2011 Keywords: Candida utilis Trehalose Trehalose-6-phosphate synthase Arginine
a b s t r a c t Background: Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6phosphate phosphatase (TPP). Methods: In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM L-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). Results: An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37 °C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl2, MgCl2 and ZnSO4, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest Vmax and lowest Km values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. General significance: Substrate specificity, Vmax and Km values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside) is an important disaccharide found widely in nature among yeast, bacteria, fungi, plants and lower animals [1]. It is accumulated during periods of nutrient starvation, desiccation and after exposure to mild heat shock [2]. The sugar serves as a stabilizer of cellular structures under stress conditions besides having an exceptional capacity to protect biological membranes and enzymes from the adverse effects of freezing or drying [3]. It also protects cells from stress induced by exposure to oxygen radicals. [1]. Recently, trehalose is reported to reduce several neural diseases by inducing autophagy including Parkinsonism, Alzheimer's disease etc. [4,5]. The trehalose pathway consists of only a few metabolites, which form a substrate cycle, and is governed
Abbreviations: TPS, Trehalose-6-phophate synthase; TPP, Trehalose phosphate phosphatases; UDPG, Uridine diphosphaphate glucose; ADPG, Adenosine di phosphate synthase; G-6-P, Glucose-6-phophate; F-6-P, Fructose-6-phosphate ⁎ Corresponding author. Tel.: + 91 33 2499 5787/5870; fax: + 91 33 2473 5197. E-mail addresses: [email protected], [email protected] (A.K. Ghosh). 0304-4165/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2011.06.025
by a surprisingly complex control system that comprises of several inhibiting or activating mechanisms [6]. The best known and most widely distributed pathway involves the transfer of glucose from uridine diphosphate glucose (UDPG) to glucose-6-phosphate (G-6-P) to form trehalose-6-phosphate (T-6-P) and uridine diphosphate (UDP) [7]. This step is catalyzed by the enzyme trehalose-6phosphate synthase (TPS). Trehalose-6-phosphate is subsequently dephosphorylated in the next step to yield inorganic phosphate and trehalose. The enzyme trehalose-6-phosphate phosphatase (TPP) catalyzes this conversion. It was reported in yeast that the enzymes involved in trehalose biosynthesis, viz. TPS and TPP exist together in the trehalose synthase complex that is highly regulated at the activity level as well as at the genetic level [8]. The trehalose synthase complex was so far purified as a single unit and the sub units could not be separated from the complex [7,8]. However, TPS was purified in its free form in our laboratory from S. cerevisiae [9]. The present report also deals with TPS purified in its free form from C. utilis. This purification excluded ammonium sulfate fractionation; instead a new methodology of cell lysis as well as HPGFLC in the presence of 100 mM arginine was introduced. L-Arginine is one of the most commonly
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used and most generally applied suppressors of protein aggregation [10]. It is frequently used as a solution additive to stabilize proteins against aggregation. The new purification protocol includes L-arginine so as to avoid the problems faced by protein aggregation phenomenon. The present investigation thus deals with purification, effects of aggregation and dependence on different factors for optimal TPS activity from C. utilis. 2. Materials and methods Trehalose-6-phosphate, uridine diphosphate glucose (UDPG), glucose-6-phosphate (G-6-P), phenylmethylsulphonyl fluoride (PMSF), benzamidine hydrochloride, 2-mercaptoethanol, glycerol, polyethylene glycol, Bradford reagent were obtained from Sigma, USA. Anthrone was procured from Aldrich, USA. Polyacrylamide gel electrophoresis reagents and chemicals were obtained from BIORAD, USA. All the other chemicals and medium components used were of analytical grade and were purchased locally.
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excluding arginine, pH 7.0. Fractions of 0.5 ml were collected and assayed for TPS activity. Active fractions were pooled and concentrated by lyophilization. 2.3.4. Re-chromatography on Biosuite 125 of Step 3 enzyme The concentrated enzyme preparation of Step 3 enzyme was passed again through Biosuite 125 with same flow rate and mobile phase as above. The active enzyme fraction was collected, pooled and stored at −20 °C for future use. 2.3.5. High performance reverse phase liquid chromatography on Delta Pak C4 The Step 4 enzyme was desalted and injected in a HPRPLC column, Deltapak C4 (300 Å, 3.9 × 300 mm, Waters, USA). Two solvents used for HPRPLC were Solvent A (Water containing 0.1% TFA) and Solvent B (Acetonitrile containing 0.1% TFA). Protein was eluted by a 60 min linear gradient to reach from 100% A to 100% B. Flow rate was maintained at 1 ml/min. A214 and A280 were monitored as stated earlier.
2.1. Organism and culture conditions 2.4. Assay of TPS activity The wild type diploid C. utilis (Cat no. NCIM/ Y500) strain used for work was the same as used in previous reports [11]. Cells were grown in YPD medium at 30 °C as stated earlier till desired stage of growth and measured by taking absorbance at 660 nm. Cells were harvested at 10,000 × g at 4–5 °C for 10 min.
TPS activity was assayed at 37 °C for 15 min using 5 mM UDPG and 5 mM G-6-P as substrates [9]. T-6-P formed was determined by anthrone color reagent after neutralizing all other sugars [12–14]. Unit of enzyme activity (U) was expressed in micromole of T-6-P synthesized per minute under assay conditions.
2.2. TPS production during growth 2.5. Protein estimation Growth curve of C. utilis strain was monitored according to the protocol mentioned in the previous reports [9]. Aliquots were collected at different hours of growth and cells were harvested by centrifugation.
Protein was measured by Bradford reagent as per technical bulletin provided by the manufacturer, Sigma. Protein content of whole cell homogenate was determined by the modified method of Lowry [15]. BSA was used as standard.
2.3. Purification of trehalose-6-phosphate synthase 2.6. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis 2.3.1. Preparation of crude extract Cells grown in YPD medium up to A660 ~ 25 were harvested, washed and suspended in ice cold lysis buffer pH 9.6 (20 mM Tris–HCl, buffer containing 1 mM EDTA, 1 mM benzamidine hydrochloride, 1 mM PMSF, 15 mM 2-mercaptoethanol, 10% (w/v) glycerol, 0.1% (w/v) Tween 40 and 100 mM arginine). The cells were lysed by passing twice through a FRENCH Pressure Cell Press (SLM Instruments, USA) at 18,000 psi and observed under microscope to confirm successful lysis. The homogenate was centrifuged at 10,000 × g for 5 min. The supernatant containing nearly 100% activity was designated as crude enzyme solution or Step 1 enzyme. 2.3.2. HPGFLC of Step 1 enzyme The crude enzyme solution from Step 1 was applied to high performance gel filtration liquid chromatography (HPGFLC) using HiLoad 16/60 Superdex 200 preparative grade column (120 ml bed volume) obtained from Amersham Biosciences, Sweden. HPLC buffer, pH 9.6 (Tris–HCl 20 mM, containing 0.1 mM Benzamidine hydrochloride, 0.1 mM PMSF, 0.5 mM 2-mercaptoethanol, 0.5% (w/v) glycerol, 0.1% (w/v) Tween 40 and 100 mM Arginine) was used as mobile phase at a flow rate of 1 ml/min. Eluted fractions were collected and A280 was measured. Fractions containing TPS activity were pooled and concentrated by polyethylene glycol (mol wt ~ 15,000–20,000 Da). 2.3.3. High performance gel filtration liquid chromatography on Biosuite 125 of Step 2 enzyme The concentrated enzyme preparation of Step 2 was passed through another HPGFLC column Biosuite 125 and monitored using dual wavelength detector, both of which have been obtained from Waters, USA, with a flow rate of 0.5 ml/min using same mobile phase but
Non denaturing PAGE of Step-4 TPS was carried out at pH 7.0 on a 7.5% polyacrylamide gel using 8 × 8 × 1.5 mm gel slab Mighty Small gel apparatus obtained from Hoefer. Electrophoresis was carried out at a constant current of 20 mA/slab and according to the guidelines in the technical bulletin provided by the manufacturer (Hoefer, USA). The movement of the proteins entirely depended on the net average negative charge exhibited in the presence of glycine (Tris–glycine buffer, pH 8.5). The single protein band was visualized on staining with Coomassie Brilliant Blue R-250 (Pierce, USA). Denaturizing sodium dodecyl sulfate-polyacryalamide gel electrophoresis (SDS-PAGE) of purified protein sample of Step 4 was carried out at pH 8.5 on a 12.5% resolving gel (8 × 8 × 1.5 mm) preparation using the same Mighty Small electrophoresis unit, Hoefer, USA and discontinuous Laemmli buffer as per GE Healthcare technical bulletin. Denatured protein bands were resolved by a constant supply of current (20 mA/slab) according to the technical bulletin provided by the manufacturer (Hoefer, USA). 2-Mercaptoethanol (5%) and ethylenediaminetetraacetic acid (EDTA; 1 mM) were added to the sample buffer as a solubilizing agent. Low molecular weight markers obtained from GE Healthcare (Code RPN 755), USA, were loaded and run in parallel lanes and gels were stained with Coomassie Brilliant Blue R250 (Pierce, USA) by shaking overnight at 37 °C. 2.7. Molecular weight estimation Molecular weight of the purified enzyme protein was estimated by HPGFLC from a plot of log of molecular weight versus Kav values of low molecular weight standards (GE Healthcare, USA). Molecular weight
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was also estimated by SDS-PAGE from a plot of Rf (relative migration values) versus log of molecular weight of LMW gel electrophoresis markers [GE Healthcare, USA]. 2.8. N-terminal sequencing The purified protein eluting out from HPRPLC was spotted on a PVDF membrane following the method suggested by the manufacturer [16]. The membrane was washed with double distilled water as per the method suggested by the manufacturer. The transferred protein bands on PVDF were stained and identified with Ponceau S solution [16]. The identified protein bands of interest were excised for N-terminal sequencing studies. The desired bands were analyzed by N-terminal sequencing using a Model 491 Procise protein/peptide sequencer from Applied Biosystems, USA. The sequence obtained from the N-terminal sequencer was matched with the database of NCBI (National Center for Biological Information) using BLAST (Basic Local Alignment Search Tool). BLAST was also used to find the similarity of the N-terminal sequence obtained here with the protein sequence of known TPS from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe. 2.9. Physicochemical characterization 2.9.1. Determination of temperature and pH optima Optimum pH was determined by measuring TPS activity at 37 °C for 15 min at different pH ranging from 5.0 to 9.5 using the following buffer systems: 0.05 M sodium acetate buffer (pH 5.0 to 5.5), 0.05 M sodium phosphate buffer (pH 6.0 to 6.5) and 0.05 M Tris–HCl buffer (pH 7.0 to 9.5). The pH corresponding to maximum activity was taken as the optimum pH. Optimum temperature was determined by measuring TPS activity at various temperatures (10 °C to 80 °C) for 15 min at pH 8.5. The temperature corresponding to maximum activity was taken as the optimum temperature. 2.9.2. Determination of temperature and pH stability For temperature stability, enzyme solution taken in 50 mM Tris–HCl buffer (pH 8.5) was pre-incubated at different temperatures (0 °C to 60 °C) for 15 min. Samples were then maintained at a temperature of 37 °C and TPS assay was performed by the addition of substrates and co-factors. TPS activities of these pre-incubated samples were measured. The stability of TPS activity was studied by keeping the enzyme solution for 30 min at 37 °C in different pH solutions ranging from pH 4.5 to 10.5. Buffers used were as follows: 0.05 M sodium acetate buffer (pH 4.5 to 5.5), 0.05 M sodium phosphate buffer (pH 6.0 to 7.0), 0.05 M Tris–HCl buffer (pH 7.0 to 9.5) and 0.05 M glycine–NaOH buffer (pH 10.5). The samples were assayed normally by adding substrates (UDPG and G-6-P) and co-factors (MnCl2 and heparin) as described earlier. TPS activities of these pre-incubated samples were measured. 2.9.3. Substrate specificity of enzyme Sugar nucleotides UDPG, ADPG, GDPG or TDPG were used as glucosyl donors and enzyme assays were carried out as described previously. In all sets, G-6-P was the universal glucosyl acceptor. In another set of experiments, UDPG was kept as the universal glucosyl donor and G-6-P, fructose-6-phosphate, (F-6-P), mannose-6-phosphate (M-6-P) or glucosamine-6-phosphate were used as glucosyl acceptors. TPS activity was represented as U/mg. 2.9.4. Requirement of polyanions for TPS activity Effect of polyanions on different glucosyl donors of TPS was determined. Assay mixtures contained 0 to 1 μg of either heparin or chondroitin sulfate or RNA along with 5 mM of G-6-P and various sugar nucleotides. Other assay components were not altered. Tris–HCl
buffer 50 mM, pH 8.5 was used as the assay buffer. Values were represented as U/mg. 2.9.5. Requirement of metal co-factors for TPS activity Activity of purified TPS was assayed at 37 °C for 15 min using 5 mM UDPG and G-6-P as substrates along with different metal salts. Assay mixture without any added metal salts was considered as the control set. Values were represented as U/mg. 2.9.6. Effect of metal chelators and inhibitors on TPS activity Purified TPS enzyme was pre-incubated with specific concentrations of different compounds in 50 mM Tris–HCl buffer (pH 8.5) for 15 min at 37 °C unless otherwise stated. Assay was started with the addition of the substrate and co-factors. Residual activities of the preincubated samples were measured. Values were expressed in percentage and TPS activity of a sample identically pre-incubated but in the absence of any compound was considered as 100%. 2.9.7. Enzyme progress curve of TPS TPS activity was measured using 0.94 μg of enzyme taken in a 100 μl of assay mixture and assayed as above. Assay mixtures were incubated for different time intervals as indicated in Fig. 7. TPS activity values were expressed as U/mg. 2.10. Regulation of trehalose-6-phosphate synthase 2.10.1. Effect of thiol modifiers on purified TPS activity Purified TPS was pre-incubated with 1 mM concentration of various thiol modifiers in 50 mM Tris–HCl buffer pH 8.5 for 15 min at 37 °C. Assay was started with the addition of the substrates and cofactors. Residual activities of the pre-incubated samples were measured. Values were expressed in percentage and TPS activity of a sample identically pre-incubated but in the absence of any chemical was considered as control. 2.10.2. TPS activity as a function of protein concentration TPS activity was measured using different concentrations of protein in 100 μl of assay mixture. Assay was done as stated above. TPS activity values were expressed as U/mg. The data obtained from the experiment of temperature stability was plotted in an Arrhenius plot and energy of activation (Ea) was calculated from the slope of the plot following a previously reported protocol [17]. 3. Results 3.1. TPS product during growth Rate of cell division increased rapidly during the exponential phase and then gradually slowed down till it plateaued during the stationary phase. TPS activity in cell lysate started increasing 10 h onwards and peaked at 36 h after inoculation when A660 values of the culture corresponded to ~25 after which it was seen to fall gradually (Fig. 1). Maximum specific activity of TPS was 0.096 U/mg protein at A660 ~ 25. 3.2. Purification of trehalose-6-phosphate synthase 3.2.1. HPGFLC of Step 1 enzyme Step 1 enzyme (cell lysate) was subjected directly to high performance gel filtration liquid chromatography (HPGFLC) using HiLoad 16/60 Superdex 200 preparative grade column from Amersham Biosciences, Sweden at a flow rate of 1 ml/min. Three protein peaks were obtained at 40, 67 and 113 min of elution (data not shown). TPS activity eluted out in the second peak (64–70 ml). The other two peaks contained no TPS activity. This was designated as step 2.
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activity 2.03 U/mg with a 22.1 purification fold and yield of 53.11% (Table 1).
3.2.5. High performance reverse phase liquid chromatography on Delta Pak C4 The Step 4 enzyme was subjected to reverse phase chromatography in a Delta Pak C4 column (Waters, USA). A sharp peak was obtained at 18.5 min (Fig. 2). The 36 min and 45 min peaks seen were obtained in blank run as well. Fig. 1. Growth curve of C. utilis stain. Growth was monitored by measuring absorbance at 660 nm (—♦—). Specific activity of TPS (—■—) was measured at different phases of growth. All experiments were conducted in triplicate.
3.2.2. High performance gel filtration liquid chromatography (HPGFLC) on Biosuite 125 of Step 2 enzyme The Step-2 TPS obtained above was concentrated by PEG and a second HPGFLC was performed with the concentrated Step-2 enzyme to analyze the effect of a second gel filtration on enzyme activity. Chromatographic profile showed a minor peak at filtration at 12.2 min and a sharp major peak at 13.8 min (data not shown). TPS activity was found to be eluted out at the second major peak from 13 to 16 min. This purification step was designated the Step-3 TPS. 3.2.3. Re-chromatography on Biosuite 125 of Step 3 enzyme The Step-3 TPS obtained above was again concentrated by PEG, and a third HPGFLC was performed to obtain a single peak with TPS activity at 13.6 min (Fig. 2). This finally purified TPS is designated as the Step 4 enzyme. 3.2.4. Purification table of TPS The purification table enumerates the four steps of the purification protocol and mentions total protein, total enzyme activity, specific activity as U/mg, purification fold and yield obtained at each purification step. Final purification step (step 4) contained specific
3.3. Native and sodium dodecyl sulfate-polyacryalamide gel electrophoresis Non denaturing gel electrophoresis of Step 4 enzyme showed a single band confirming the purity of the enzyme (Fig. 3A). SDS PAGE analysis of Step 4 enzyme showed a single band and which was calculated as 60 kDa when compared with standard protein markers (LMW gel electrophoresis markers) thereby confirming the purity of the enzyme (Fig. 3B).
3.4. N-terminal sequencing A 20 residue long amino acid sequence of the N-terminal of the protein was obtained as ‘DTVESEIAFVFDDLGEEXFF’. BLAST analysis with NCBI database revealed that none of the non redundant protein sequences matched completely with the sequence obtained from the N-terminal sequencing which was expected since no TPS sequence from C. utilis is available yet. BLAST analysis was also done to match the sequence with known protein sequences of TPS from related species of yeast which revealed that the experimentally obtained N-terminus of the purified protein was most similar to TPS from S. cerevisiae followed by TPS from S. pombe (Table 2). The alignment of the N-terminal sequence with these two sequences was also matched using EMBOSS Pairwise Alignment Algorithm Needle.
Fig. 2. Step 5: Re-chromatography on Biosuit 125 and high performance reverse phase liquid chromatography on Delta Pak C4. Enzyme solution obtained from step 3 was separated by using HPGFLC column Biosuit 125 as described in the text. Enzyme solution obtained from step 4 was separated by using HPRFLC column Delta Pak C4 as described in the text.
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Table 1 Purification table of TPS from C. utilis. Steps
Total protein (mg)
Total enzyme activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
Soluble supernatant (Step 1) HPGFLC on Hiload 16/60 Superdex 200 (Step 2) HPGFLC Biosuite 125 (Step 3) HPGFLC Biosuite 125 (Step 4)
183.2 ± 0.81 12.1 ± 0.76 4.1 ± 0.61 2.9 ± 0.33
17.6 ± 0.31 13.9 ± 0. 26 7.4 ± 0.22 5.9 ± 0.17
0.096 ± 0.001 1.14 ± 0.002 1.804 ± 0.09 2.03 ± 0.07
1 11.97 18.84 22.192
100 74.25 64.22 53.11
Purification was performed as described in the text. Values represented are average of three sets of purification data. The standard deviation of each measurement is indicated.
3.5. Determination of temperature and pH optima The pH optimum was recorded at pH 8.5 with 1.97 U/mg activity (data not shown). TPS activity was seen to be more affected at low pH conditions. The Step-4 TPS enzyme displayed a classical bell curve of temperature optima. Highest TPS activity was observed at 37 °C with 1.99 U/mg activity. Very low activities of 0.4 U/mg were detected at 10 °C and 60 °C beyond which no activity of TPS was seen (data not shown). 3.6. Determination of temperature and pH stability The Step 4 enzyme activity was more or less stable in the temperature range of 0–40 °C (data not shown). It showed a gradual decrease in stability with increasing temperature. Even at 60 °C, around 40% activity was retained. TPS activity appeared to be maximally stable at pH 8.5 with 2.11 U/ mg activity, followed by 2.01 U/mg activity at pH 9.0 and 1.93 U/mg activity at pH 9.5 (data not shown). In the highly alkaline condition of pH 10.5, 1.21 U/mg activity was observed. Stability of TPS activity was more affected at lower pH conditions. Only 0.31 U/mg activity was retained at pH 4.5. 3.7. Substrate specificity of TPS TPS activity was assayed using different sugar nucleotides to observe whether they could act as potential glucosyl donors for the purified enzyme. Maximum TPS activity was obtained with ADPG (~3.5 U/mg), while with UDPG, the activity was nearly halved to 1.9 U/mg (Fig. 4A). GDPG gave 0.9 U/mg activity while TDPG showed the least effect on TPS by displaying the lowest activity (0.6 U/mg) among the four. Substrate specificity of TPS with respect to its glucosyl
acceptor displayed a specific activity of ~ 2.6 U/mg with G-6-P while F-6-P displayed an activity of ~ 1 U/mg which was ~ 2.5 times less the activity obtained with G-6-P (Fig. 4B). M-6-P and Glucosamine-6-P were not glucosyl acceptors for TPS activity. The initial velocity (v) of the enzyme activity was seen to increase gradually with increasing concentration of UDPG and G-6-P from 0.5 mM to 20 mM. Km value for ADPG was determined as 1.36, while those for UDPG, GDPG and TDPG were observed to be 2.15, 2.24 and 2.81 respectively (Table 3). Lineweaver Burk's plots displayed Vmax of purified TPS for different glucosyl donors such as 2.96 for ADPG, 2.41 for UDPG, 2.37 for GDPG and 2.35 and TDPG (Table 3). Km and Vmax values were different from those obtained from TPS from S. cerevisiae [18]. Km values for S. cerevisiae TPS is 2 mM for UDPG since ADPG is not the substrate [18]. 3.8. Requirements of metal co-factors for TPS activity Assay mixtures contained 5 mM of respective metal salts as mentioned in Table 4. Assay mixture without any added metal was considered as the control set. TPS activity was markedly stimulated with 5 mM MnCl2, and showed maximum activity of 1.513 U/mg. The enzyme was relatively specific for MnCl2, and was much less active in case of other metals. Most metals used were not able to replace MnCl2, or were only slightly effective in this regard. MgCl2, at 5 mM concentration, was also effective and displayed TPS activity of 1.47 U/mg. Optimum concentration in case of both MnCl2 and MgCl2 was seen to be 70 mM (Fig. 5). TPS activity in the presence of ZnSO4 and CuSO4 displayed greater activity than that of control. Several metals were seen to display lower activity than that of control, suggesting a potential inhibition of enzyme activity. These metals included CdCl2 and AgNO3 being the most potent. 3.9. Dependence on polyanion concentrations for optimum TPS activity Heparin, chondroitin sulfate and RNA were used as polyanions. Assay mixtures contained heparin from 0 to 1 μg along with 5 mM of G-6-P and different sugar nucleotides UDPG, GDPG, ADPG and TDPG. As observed previously, ADPG displayed maximum TPS activity with heparin. ADPG was observed to be the best substrate, and displayed maximum activity of 4.23 U/mg at 0.15 μg heparin, followed by UDPG (2.016 U/mg) and GDPG (1.609 U/mg). TDPG displayed maximum activity of 0.95 U/mg at 2 μg (Fig. 6A). TPS activity fell after 0.20 μg heparin concentration. Similar results were observed with
Table 2 Summary of the BLAST analysis.
Fig. 3. Gel electrophoresis: SDS and native. Non denaturing PAGE and SDS PAGE were carried out as described in the text. SDS PAGE of step 4 enzyme and low molecular weight markers were run. Protein standards were from the LMW calibration kit from Amersham Biosciences, UK. Corresponding molecular weights are indicated next to the protein bands. Marker lanes in SDS PAGE were considered for molecular weight estimation.
Accession no.
Organism
Max score
Total score
Query coverage (%)
E value
ADM26405
Saccharomyces cerevisiae Schizosaccharomyces pombe Candida albicans
16.3
41.4
28
0.14
11.2
19.5
53
4.8
10.8
31.8
35
6
P40387 Q92410
N-terminal sequence obtained was matched with the database of NCBI. Sequences were matched using BLAST (Basic Local Alignment Search Tool). Details of the matches, TPS from S. cerevisiae, C. albicans and S. pombe are given.
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Table 4 Effects of cofactors on TPS activity. Metal
Concentration (mM)
Specific activity (U/mg)
Activity (%)
None MnCl2 MgCl2 ZnSO4 CuSO4 CoCl2 CaCl2 BaCl2 FeSO4 CdCl2 AgNO3
– 5 5 5 5 5 5 5 5 5 5
0.589 ± 0.2 1.513 ± 0.22 1.47 ± 0.17 1.314 ± 0.19 0.786 ± 0.32 0.641 ± 0.35 0.631 ± 0.3 0.567 ± 0.16 0.524 ± 0.21 0.461 ± 0.22 0.32 ± 0.23
100 256.87 249.57 223.1 133.45 108.83 107.13 96.265 88.96 78.27 54.33
TPS assay was done as described in the text. Sets containing purified TPS were preincubated for 15 min at 37 °C. All experiments were performed in triplicate. Standard deviation of each measurement is indicated.
3.12. Regulation of trehalose-6-phosphate synthase
Fig. 4. Substrate specificity of TPS. Enzyme assays were performed as described in the text. (A) Sugar nucleotides ADPG, UDPG, GDPG and TDPG were used as glucosyl donor. (B) Sugar phosphates G-6-P, F-6-P and M-6-P were used as glucosyl acceptor. In both cases, white bars denote enzyme activity in U/mg. Data given are the average of three sets of values, which did not vary more than ±5% represented by error bars.
chondroitin sulfate as well (Fig. 6B). An increase in TPS activity was seen up to 0.2 μg post which fell with increasing concentration. ADPG displayed the highest activity of 3.8 U/mg followed by UDPG (2.8 U/ mg). GDPG and TDPG displayed activity values of 2.1 U/mg and 1.1 U/ mg respectively. RNA had no effect on the activity of TPS.
3.12.1. Effect of thiol modifiers on TPS activity TPS activity was seen to enhance in the presence of IAA and IAM (Table 6). When 1 mM concentration of IAA was used, TPS was observed at 147% residual activity. IAM also enhanced TPS activity displaying 139% of residual activity. However, when 1 mM NEM was used, TPS activity was inhibited resulting in only 32.41% of residual activity. 3.12.2. TPS activity as a function of protein concentration TPS activity was measured using different concentrations of protein in 100 μl of assay mixture. Activity could only be detected when protein concentration was increased to 0.2 μg of protein. No activity was detected when protein concentration was less than 0.2 μg. When 0.50 μg protein was used, increase in specific activity was seen. Further increasing protein concentration to 1 μg protein resulted in the highest specific activity of TPS. However, specific
3.10. Effect of metal chelators and inhibitors on TPS activity Purified TPS was pre-incubated with different metal chelators and inhibitors (Table 5). Some of the metal chelators were seen to increase TPS activity over control like EGTA and CGTA with residual activity as 129% and 141% respectively. Citric acid and EDTA were seen to inhibit TPS activity as they were observed to display 79% and 80% residual activity.
3.11. Enzyme progress curve of TPS An enzyme progress curve was prepared using different incubation time. TPS activity was seen to maintain linearity from 3 min to 15 min of incubation time after which the linearity was lost (Fig. 7). Table 3 Effect of substrate concentration on reaction velocity. Glucosyl donors
Vmax (U/mg)
Km (mM)
ADPG UDPG GDPG TDPG
2.96 ± 0.07 2.41 ± 0.09 2.37 ± 0.01 2.35 ± 0.08
1.36 ± 0.08 2.15 ± 0.01 2.24 ± 0.02 2.81 ± 0.08
Michaelis constant (Km) and maximum velocity (Vmax) for TPS activity measured at increasing concentration of ADPG, UDPG, GDPG and TDPG keeping G-6-P concentration constant at 5 mM. Data given are average of three sets of values. The standard deviation of each measurement is indicated.
Fig. 5. Effect of MgCl2 and MnCl2 on TPS activity. (A) Effect of MgCl2 on TPS activity was studied using 0, 10, 20, 50, 75 and 100 mM of the salt. (B) Effect of MnCl2 on TPS activity was studied using 0, 10, 20, 50, 75 and 100 mM of the salt. Assays were performed as described in the text. Data given are the average of three sets of values, which did not vary more than ± 5% represented by error bars.
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Fig. 7. Enzyme progress curve of TPS. Enzyme assays were performed as described in the text but with different incubation times. Assay mixture reactions were stopped at different time intervals of 0, 3, 5, 10, 15, 20, 30, 35 and 60 min. Data given are the average of the three sets of values, which did not vary more than ±5% represented by error bars.
Fig. 6. Effect of polyanions on different glucosyl donors. Enzyme assays were performed as described in the text. (A) Heparin at different concentrations of 0, 0.05. 0.1, 0.15, 0.2, 0.5 and 1 μg were taken. Glucosyl donors used were ADPG (—♦—), UDPG (—■—), GDPG (—●—) and TDPG (—▲—). (B) Chondroitin sulfate at different concentrations of 0, 0.05. 0.1, 0.15, 0.2, 0.5 and 1 μg were taken. Glucosyl donors used were the same as for heparin. Data given are the average of three sets of values, which did not vary more than ± 5% represented by error bars.
activity decreased beyond this point on further increasing protein concentration (data not shown). An Arrhenius plot of this enzyme showed a sharp discontinuity after 40 °C. Activation energy at 5 to 40 °C was calculated to be 35.65 cal/mol. At temperature 50 to 60 °C, activation energy was calculated as 7885.6 cal/mol (Fig. 8). 4. Discussion Trehalose is a multifunctional sugar which is biosynthesized in S. cerevisiae during the beginning of the stationary phase [9,19]. Since the synthesis of sugar is catalyzed by the enzymes trehalose-6phosphate synthase (TPS) and trehalose-6-phoshate phosphatases (TPP), the level of trehalose in the cell is dependent on the activity profiles of these enzymes. The present study reports purification and characterization of only TPS in its free form from C. utilis. Growth curve of C. utilis showed that the TPS activity was highest at A660 ~ 25 which was obtained at 36 h of growth, after which it was seen to fall gradually (Fig. 1). Thus, all the purification experiments of TPS were initiated at A660 ~ 25. TPS purified from S. cerevisiae attained the highest level at 33 h when A660 ~ 18 [9].
Table 5 Effect of chelators and inhibitors on TPS activity. Serial Chemical added
Conc. (mM) Activity (U/mg) Residual activity (%)
1. 2. 3. 4. 5. 6. 7. 8.
– 2.5 2.5 2.5 2.5 2.5 2.5 2.5
None CDTA EGTA 5′-sulfosalicylic acid Tri sodium citrate Sodium azide EDTA Citric acid
1.45 ± 0.11 2.05 ± 0. 19 1.88 ± 0.14 1.527 ± 0.13 1.423 ± 0.23 1.17 ± 0.22 1.16 ± 0.14 1.15 ± 0.19
100 141.25 129.56 105.31 98.12 81.21 80.15 79.14
TPS assay was done as described in the text. Sets containing TPS were pre-incubated for 15 min at 37 °C. All experiments were performed in triplicate. The standard deviation of each measurement is indicated.
Many reports of TPS purification from various organisms are reported to date. Londesborough and Vuorio obtained a 3 fold purification of TPS from S. cerevisiae [7]. TPS was also purified by Vandercammen et al. with 40 fold purification from S. cerevisiae [20]. 105 fold purified TPS has been reported from a basidiomycetous fungus Grifola frondosa [21]. Earlier reports from our laboratory reported the isolation of TPS enzyme from the trehalose synthase complex in its free form from S. cerevisiae for the first time [9]. Here, we have reported the purification of TPS from C. utilis for the first time by using a new purification strategy. A high purification fold of 22.1 was achieved as compared to 15.8 purification fold reported previously from our laboratory (Table 1) [9]. This makes this purification strategy better than the previous one reported from our laboratory. The purification process excluded ammonium sulfate fractionation, and instead introduced a new methodology of cell lysis as well as HPGFLC in the presence of 100 mM arginine. When purification of TPS was performed with the traditional protocol it resulted in a protein peak at 12.1 min during final HPGFLC which showed reduced enzyme activity and subsequent degradation with time (data not shown). This observation might be due to proteolysis or a protein aggregation phenomenon. Thus, to avoid this consequence, arginine was introduced in cell lysis as well as HPGFLC buffer. Arginine is known to have an anti-aggregation property and its role as an aggregation suppressor has been reported in detail [10]. Hence, the presence of 100 mM arginine in the initial purification step resulted in a shift of the protein peak to 13.6 min with stable activity during final HPGFLC (Fig. 2). This also resulted in improved yield due to suppressed aggregation as evident from the protein peak shift. A single band was visualized in both native and SDS PAGE confirming the purity of the Step 4 enzyme. The molecular weight of the enzyme was calculated as 60 kDa (Figs. 2 and 3). To confirm the identity of the purified protein as TPS, N-terminal sequence analysis of the 60 kDa band was performed. A 20 residue long sequence was obtained from the N terminus and this displayed various levels of similarity with different known sequences of TPS obtained from different yeasts. Around 53% of query coverage and 4.8 E value were obtained with S. pombe (Table 2). 35% query coverage
Table 6 Effect of thiol modifiers on purified TPS activity. Thiol modifiers
Conc (mM)
Activity (U/mg)
Residual activity (%)
None (control) Iodoacetic acid Iodoacetamide N-ethylmaleimide
0 1.0 1.0 1.0
1.45 ± 0.3 2.14 ± 0.32 2.02 ± 0.27 0.47 ± 0.21
100 147.5 139.3 32.41
TPS assay was done as described in the text. Sets containing TPS were pre-incubated for 15 min at 37 °C. All experiments were performed in triplicate. The standard deviation of each measurement is indicated.
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Fig. 8. TPS activity as a function of temperature (Arrhenius plot). Activity was measured as stated in the Materials and methods with different temperatures (5–60 °C). Values were obtained from the average of three sets of triplicates. The activation energy (Ea) values (cal/mol) were calculated from the slope of the plots and are shown in the inset.
and 6 E-value were obtained with C. albicans and 28% query coverage with S. cerevisiae and lowest E-value of 0.14 showed its maximum resemblance with the purified TPS from S. cerevisiae. Since comparison of 20 amino acid residues showed more resemblance with the TPS sequence from S. cerevisiae, it was supposed that the 60 kDa band which had been purified and isolated from the complex was indeed TPS. A different physicochemical characterization of the purified TPS was performed to obtain a better idea of the enzyme. The pH optimum of TPS from C. utilis was similar to that of TPS from S. cerevisiae and TPS from M. smegmatis (data not shown) [18,12]. The temperature optimum of TPS from C. utilis showed a similarity with that of TPS from S. cerevisiae [18] and basidiomycetes fungus G. frondosa [21] (data not shown). The C. utilis TPS was stable at 40 °C for 15 min, in comparison to G. frondosa TPS, where nearly all activity was lost after 30 min incubation at 40 °C [21]. This observation was similar to TPS from S. cerevisiae [18]. Purified C. utilis TPS was also observed to be analogous to S. cerevisiae and G. frondosa TPS in terms of their pH stability, as both demonstrated more stability at alkaline pH [18,21]. After the identification of the purified enzyme as TPS, different possible substrates of the enzyme were identified other than the ubiquitous UDPG and G-6-P. Substrate specificity experiments were performed both for glucosyl donors as well as acceptors, and varying levels of activity with different substrates were obtained. This study showed a remarkable observation where TPS activity was at the maximum with ADPG than its usual substrate UDPG (Fig. 4). The same enzyme also showed activity with GDPG and TDPG. TPS with ADPG as a substrate showed high Vmax and a low Km value confirming it to be the ideal substrate for this enzyme (Table 3). TPS from S. cerevisiae also showed activity with different glucosyl donors except ADPG, which was not a substrate [18]. Thus, these observations provided an insight into an altered trehalose pathway occurring in C. utilis using ADPG as a substrate instead of UDPG. Similar observations are reported in some mutant strains of S. cerevisiae where ADPG dependent trehalose synthesis pathway is present [22]. Since in these mutants the normal trehalose synthesis pathway is disrupted, trehalose synthesis via ADPG dependent trehalose synthase was preferred [22]. In the present study, pure TPS was able to carry out independent conversion of F-6-P to G6-P. It was known that TPS had some associated isomerase activity and is reported to be a bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI) from S. cerevisiae [18,23]. Metal ions function as enzyme co-factors either by binding to the actual catalytic site of the enzyme or by converting the enzyme into its active form without being directly involved in catalysis [24]. Previous reports showed that the requirement for metal co-factors was not mandatory but the presence of some metals like MnCl2 and ZnCl2 enhanced TPS activity in S. cerevisiae [18]. In the present report also, the effects of co-factors has been established as not mandatory for TPS activity (Table 4). However, the presence of some metals such as
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MnCl2 and MgCl2 enhanced TPS activity. There are reports of MnCl2 as a co-factor of TPS activity from M. smegmatis [12]. Metals like CdCl2, FeSO4, AgNO3 and BaCl2 were possible inhibitors to enzyme activity, as they displayed lower activity than control. Though MnCl2 and MgCl2 were seen to enhance TPS activity, it was, however, observed that both of them caused inhibition of enzyme activity at a high concentration (100 mM) (Fig. 5). A high MnCl2 concentration possibly caused charge re-distribution in the enzyme molecule, resulting in separation of enzyme subunits and decrease in activity [25]. There are several reports of heparin and other associated polyanions affecting TPS activity in mycobacteria as well as S. cerevisiae [14,18]. In the present report the effect of heparin was most prominent with ADPG. A similar pattern was also observed in the case of UDPG followed by GDPG and then TDPG (Fig. 6A). Similar results were obtained when chondroitin sulfate was used instead of heparin (Fig. 6B). These observations are not analogous to the pattern followed in S. cerevisiae where effects of the polyanions were more predominant on UDPG [18]. Activity of TPS was increased in comparison with control in the presence of metal chelators like EGTA and CDTA. The possible explanation behind such an observation might be that these two are specific chelators of those metals which may be potential inhibitors of TPS (Table 5). In contrast, TPS activity deceased in the presence of non specific metal chelators like EDTA, sodium azide and citric acid. Thus, non specific chelation of potential stimulators for TPS possibly resulted in reduced activity upon incubation. These observations were in accordance to S. cerevisiae TPS [18]. Enhancement of enzyme activity by IAA and IAM posed to be an interesting study (Table 6). IAA acts by incorporating an acetic acid group in cysteine and causes carboxymethylation, while IAM incorporates an acetamide group in the amino acid to cause carboxyamidomethylation [26]. NEM modifies the thiol group of cysteine by incorporating a bulky group that does not cause any carboxymethylation [26]. Contrary to IAA and IAM, NEM was seen to inhibit TPS activity. Hence, it appeared that effects of IAA and IAM on TPS were carboxymethylation related. This observation was similar with TPS from S. cerevisiae where TPS activity was enhanced due to carboxymethylation of cysteine residues [18,27]. When protein concentration was increased up to 1 μg TPS activity was seen to increase. However specific activity decreased when protein concentration was further increased. At 2.5 μg protein concentration a ~ 30% decrease in the TPS activity was observed. This showed that increasing the concentration of reacting molecules caused an increase in specific activity up to a certain concentration. It thus appeared that aggregation of protein was important for regulation of TPS activity. TPS showed linearity up to 15 min of incubation time after which linearity was lost (Fig. 7). When TPS was subjected to different temperature changes a sharp discontinuity was observed in the Arrhenius plot corresponding to a large change in the activation energy (Fig. 8). A sudden drop in the Arrhenius plot indicated enzyme deactivation which is due to protein aggregation phenomenon [28]. When an enzyme exhibits an aggregation–de-aggregation state having a different activation energy, then a sharp discontinuity is observed. Since activation energy at 40–60 °C is much higher than that at 5–40 °C, it is expected that at a higher temperature change in enthalpy (ΔH) or entropy (ΔS) will be higher. Hence, the presence of de-aggregated form is expected at a higher temperature and aggregation is favored at a lower temperature. This kind of an identical study is also reported in xylanolytic amyloglucosidase [17]. 5. Conclusion It can be concluded that a new purification strategy is introduced during purification of TPS from C. utilis. This purified TPS exhibited an altered trehalose metabolic pathway using ADPG and G-6-P as
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