Role of a disulphide bond in Helicobacter pylori arginase

Biochemical and Biophysical Research Communications 395 (2010) 348–351

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Role of a disulphide bond in Helicobacter pylori arginase Abhishek Srivastava, Nidhi Dwivedi, Apurba Kumar Sau * National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India

a r t i c l e

i n f o

Article history: Received 16 March 2010 Available online 8 April 2010 Keywords: H. pylori arginase Disulphide bond Thermal stability Co(II) CD

a b s t r a c t Arginase is a binuclear Mn2+-metalloenzyme of urea cycle that hydrolyses arginine to ornithine and urea. Unlike other arginases, the Helicobacter pylori enzyme is selective for Co2+. Previous study reported that DTT strongly inhibits the H. pylori enzyme activity suggesting that a disulphide bond is critical for the catalysis. In this study, we have undertaken steady-state kinetics, circular dichroism and mutational analysis to examine the role of a disulphide bond in this protein. By mutational analysis, we show that the disulphide bond is not important for catalytic activity; rather it plays an important role for the stability of the protein as observed from thermal denaturation studies. The loss of catalytic activity in the wildtype protein with DTT is due to the interaction with Co2+. This is verified with the Mn2+-reconstituted proteins which showed a marginal loss in the activity with DTT. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Arginase, a binuclear Mn2+-metalloenzyme of urea cycle converts arginine to ornithine and urea [1]. Helicobacter pylori (H. pylori) is a human pathogen which causes peptic ulcer disease, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma [2–4]. H. pylori has a complete urea cycle and contains the rocF gene encoding arginase [5–7]. This enzyme produces endogenous urea that can be utilised by urease to generate ammonia and carbon dioxide, and thus helps in neutralising the surrounding acidic environment so that the bacterium can survive. The rocF gene in H. pylori has been shown to be moderately important for colonisation in the gastric epithelial cells [8]. It also inhibits host nitric oxide production by activated macrophage at physiological concentration of arginine [9]. A higher level of NO production was found by macrophages when rocF-deficient H. pylori was co-cultured resulting in efficient killing of the bacteria, whereas the wild-type bacteria exhibited no loss of survival [9]. It plays a role in inhibiting T cell proliferation by reducing expression of CD3f [10]. In contrast to other arginases, the H. pylori enzyme shows several unique features such as optimal activity with Co2+ instead of Mn2+ as a metal cofactor, highest activity at low pH (6.1 instead of 9.5), presence of a unique motif with 13 residues and considerable differences at the N- and C-terminal sites [7]. This suggests that there could be a difference in the active site architecture compared to the other arginases. Thus, it may be an important target for the development of new antibiotics. An intact bimetallic site

* Corresponding author. Fax: +91 11 26742125/26742626. E-mail addresses: [email protected], [email protected] (A.K. Sau). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.014

at the active site is essential for catalysis in all arginases. A water molecule bridged between the two metal ions has been suggested to be important for the nucleophilic attack to guanidine group [11]. Previous studies reported that the H. pylori enzyme lose activity in the presence of a reducing agent DTT in a dose dependent manner [7]. It was suggested that the disulphide bond plays an important role in the activity of the protein. Mass spectrometric studies revealed that the H. pylori enzyme contains a disulphide bond between Cys66 and Cys73 [12]. It has been hypothesised that the disulphide bond in the H. pylori enzyme may have a unique role in activity, as mammalian and other bacterial arginases are resistant to DTT. To examine the role of this disulphide bond, we carried out mutagenesis, steady-state kinetics and circular dichroism studies. We mutated the Cys residues individually to Ala and examined their role in the catalysis and stability. We also investigated the role of the metal ions in the stability of the wild-type and mutant proteins. Surprisingly, our studies show that both mutants Cys66Ala and Cys73Ala were catalytically active indicating that the disulphide bond is not important for the activity. Rather, it plays an important role in the stability of the protein as observed from the thermal denaturation studies. Furthermore, a mechanism for the loss of the wild-type activity with DTT is discussed. 2. Materials and methods 2.1. Cloning and mutagenesis The wild-type gene was cloned according to the protocol as described in the unpublished data. Cys66Ala and Cys73Ala mutants of H. pylori arginase were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, USA) according to the

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A. Srivastava et al. / Biochemical and Biophysical Research Communications 395 (2010) 348–351

2.2. Protein expression and purification The wild-type and mutant proteins were transformed into Escherichia coli BL21 (DE3) competent cells. The proteins were over-expressed and purified following a procedure as described in the unpublished data. The protein was concentrated and stored at 80 °C with 10% glycerol. The concentrations of the mutant proteins were determined spectrophotometrically using a Bio-Rad assay. After purification of the proteins, the GST-tag was cleaved using a protease enzyme [13]. 2.3. DTNB assay DTNB (5,50 -dithio-bis(2-nitrobenzoic acid)) (Pierce, USA) also known as Ellman’s reagent [14] reacts with free sulfhydryl groups resulting in formation yellow thio-nitrobenzoate anion (TNB2) which absorbs at 412 nm. The reaction was started by mixing the protein with 250 mM Tris (pH 8.0) buffer containing the Ellman’s reagent at 25 °C. The buffer was degassed before usage. The concentrations of DTNB and arginase were 250 and 20 lM, respectively. The resulting formation of the yellow thio-nitrobenzoate anion (TNB2) was followed by monitoring the absorbance at 412 nm [14] using a Shimadzu UV-2450 UV–vis spectrophotometer. The number of reacted cysteines was calculated from the absorption values at 412 nm [15]. 2.4. Activity assay The arginase assay was carried out spectrophotometrically by measuring the formation of ornithine at 515 nm with ninhydrin at around pH 5.0 [7]. The assay buffer containing metal ions was pre-incubated with apo-arginase for 30 min at 50 °C to activate the enzyme. The mixture was brought down to room temperature and the reaction was finally carried out at 37 °C by adding arginine. The reaction was stopped with 375 ll of glacial acetic acid and the colour was developed with the addition of 125 ll ninhydrin (4 mg/ ml) at 90 °C for 1 h. A standard curve with different dilutions of known concentration of ornithine was generated and slope of the curve was determined. This slope was used to measure the formation of ornithine in the reaction mixture. A control was always used to subtract the background in each concentration of arginine. 2.5. Circular dichroism Circular dichroism (CD) measurements of the wild-type and mutant proteins in the presence of different metal ions were done on a Jasco-810 spectropolarimeter. Prior to measurements the reaction mixture was incubated at 50 °C for about 10 min and then the measurements were carried out at 25 °C. The optical cell of 1 mm path length was used for all measurements. All spectra were corrected by subtracting the baselines of buffer and buffer plus metal ion, recorded under the same condition. All measurements were carried out using 20 mM Tris, pH 7.4.

pylori enzyme contains a disulphide bond (Cys66–Cys73) and it is believed to be important for the catalytic activity, since the protein loses activity upon addition of a reducing agent DTT. To examine the role of this disulphide bond in catalysis, we made individual mutants Cys66Ala and Cys73Ala. We first employed DTNB assay using Ellman’s reagent for the wild-type as well mutant proteins to estimate the number of the free sulfhydryl group. In this assay, DTNB (5,50 -dithio-bis(2-nitrobenzoic acid)) reacts with free cysteine residues that are not involved in the disulphide bond formation. By concomitant formation of stoichiometric amounts of the yellow thio-nitrobenzoate anion (TNB2), the number of reactive cysteine residues can be defined and the time course of their oxidation can be monitored. As observed in Fig. 1, the number of cysteine residues in the wild-type protein was four, which is in good agreement with the earlier report. It is also clear from the time course experiment (Fig. 1) that three residues are freely accessible to solvent, whereas one is relatively less solvent exposed. We carried out similar assays with Cys66Ala and Cys73Ala mutants. Analysis of the data showed that the mutant proteins contain approximately five cysteine residues, consistent with Cys66– Cys73 forming a disulphide bond in the wild-type protein. To examine the role of the disulphide bond, we carried out steady-state kinetic assays of the wild-type as well as mutant proteins Cys66Ala and Cys73Ala. In these assays, the substrate was used in excess over the enzyme so that multiple turnovers can be examined. As shown in Table 1, the catalytic turnover (kcat) and the catalytic efficiency (kcat/Km) for the mutant proteins showed similar results to the wild-type suggesting that the disulphide bond does not play a significant role in the catalysis. To investigate how DTT affects the activity of the wild-type protein, we carried out similar assays with the Co2+-reconstituted wild-type as well as mutant proteins in the presence of DTT. Interestingly, both the wild-type and mutant proteins show loss of activity with DTT in a concentration dependent manner with Co2+ as a metal cofactor (Fig. 2). This suggests that the loss of activity in these proteins could be due to the interaction of DTT with Co2+ ions. It has been reported that the reducing agents containing sulphur group such as DTT and b-mercaptoethanol have stronger affinities for the metals Co2+, Ni2+, Cd2+ and Hg2+ [16,17]. DTT perhaps forms a complex with Co2+ so that the free metal ion is not available to form holoprotein. To investigate whether this is specific for Co2+, we carried out similar assays with Mn2+-reconstituted protein with and without DTT. Interestingly, as observed in Fig. 2 Mn2+-reconstituted

5 Number of Cysteine Residues

manufacturer’s protocol. Appropriate forward and reverse primers with GST-tagged pC6-2-rocF plasmid as a template were used for PCRs. The positive mutants were then identified by DNA sequencing.

4

3

2

1

Wild Type Cys73Ala Cys66Ala

0 0

3. Results and discussion 3.1. Disulphide bond is not important for catalysis Analysis of the sequence of H. pylori arginase reveals that this protein contains six cysteine residues. As described earlier, the H.

10

20

30

40

50

60

Time (min) Fig. 1. Reaction of arginase with DTNB. The formation of yellow thio-nitrobenzoate anion (TNB2) was monitored at 412 nm and was converted into equivalent reacted cysteine residues. Time course reaction was carried out and total number of free cysteine residues obtained in the wild-type, Cys73Ala and Cys66Ala were 3.8 (4), 5 and 4.5 (5), respectively.

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Table 1 Steady-state kinetic parameters of the wild-type and mutant H. pylori arginases at 20 mM Tris–HCl, pH 7.2. The experiments were carried out by taking a fixed concentration of pre-incubated reaction mixture containing apo-enzyme and metal with varying concentrations of arginine as described in Section 2. The apparent Km, kcat and n (Hill co-efficient) were analysed using Hill equation, Rate ¼ kcat  ½E0   ½Argn =ðK nm þ ½Argn Þ. The quality of fit was judged by a theoretical line drawn through the experimental data points and highest confidence limit.

Wild-type Cys66Ala Cys73Ala

kcat (min1)

Km (mM)

kcat/Km (mM1 min1)

n

31 22 32

6.2 6 5

5 3.7 6.4

1.8 2.3 2.5

1

A

0 -1 CD(mdeg)

350

-2 -3 -4

Tm (°C)

Cys73Ala

Apo

Holo

Apo

Holo

Apo

Holo

69

71

62

66

62

67

Mn Co

A 100 80

1.0

220

230

240

250

B

0.8 0.6 0.4

60

0.2

40

0.0

Apo WT Holo WT

30

20

40

50

60

70

80

90

T ( 0C) Fig. 3. (A) Circular dichroism measurements of the wild-type and mutant proteins with metal ions. Both wild-type and mutant proteins were heat activated. In heat activation step, the samples were kept at 50 °C for about 10 min and then the measurements were done at 25 °C. The concentration of the proteins and Co2+ was 1.5 and 30 lM, respectively. The data were collected before the saturation of high voltage. (B) Thermal denaturation of the wild-type arginase by circular dichroism measurements. The protein was heated from 30 to 90 °C, and their denaturation was followed by molar ellipticity at 220 nm. The fraction denatured at each temperature was determined and plotted. Similar experiments were carried out for mutants Cys73Ala and Cys66Ala. Melting temperature of the wild-type and mutant proteins is shown in Table 2.

120 0

B 100 80 % Activity

210

Wavelength (nm)

120

% Activity

200

Cys66Ala

Fraction Denatured

Wild-type

Wild type Cys66Ala Cys73Ala

-5

Table 2 Melting temperature (Tm) of the wild-type and mutant proteins with and without Co2+ ions.

60 40 20 0 0

100

200 300 DTT (µM)

400

500

Fig. 2. (A) Activity assays of Co2+- and Mn2+-reconstituted Cys73Ala mutant proteins in 20 mM Tris–HCl, pH 7.2, and in presence of DTT. The assay was carried out with a fixed concentration of 1 lM apo-enzyme and the metal ions with varying concentrations of DTT. The metal-reconstituted enzyme was heat activated at 50 °C for about 30 min and the assay carried out by adding arginine at 37 °C. (B) Activity assays of Co2+- and Mn2+-reconstituted wild-type enzyme in 20 mM Tris–HCl, pH 7.2, and in presence of DTT. The assay was carried out with a fixed concentration of 1 lM apo-enzyme and the metal ions with varying concentrations of DTT. The metal-reconstituted enzyme was heat activated at 50 °C for about 30 min and the assay carried out by adding arginine at 37 °C.

protein showed a marginal loss of activity (20%) with DTT compared to Co2+. The mutant proteins showed similar result to the

wild-type confirming that the loss of activity with DTT is due to the interaction with Co2+ ions. It is also known that sulphur group has lower affinity for Mn2+ compared to Co2+. This agreed well with histidine, aspartate and glutamic acids being preferred ligands for Mn2+-proteins [18,19]. This also explains why mammalian and other bacterial arginases are not sensitive to DTT with Mn2+ as a metal cofactor. 3.2. Disulphide bond is important for the stability To understand whether the disulphide bond has any role in the overall secondary structure of the protein, we carried out circular dichroism (CD) measurements of the wild-type and mutant proteins with and without metal ions. The CD spectrum of the apoproteins showed minima at 208 and 222 nm, which is indicative of a helical protein. The CD spectra of the wild-type holo and apo-proteins are similar indicating that the metal ions do not have significant role in the secondary structure of the wild-type protein (data for apo-protein is not shown). Cys73Ala showed results similar to the wild-type indicating that the disulphide bond does not

A. Srivastava et al. / Biochemical and Biophysical Research Communications 395 (2010) 348–351

play an important role in the overall secondary structure of the protein (Fig. 3A). Cys66Ala showed a marginal loss in the structure in the presence of the metal ions compared to the wild-type. To investigate whether the disulphide bond is important for the stability of the protein, the heat-induced denaturation curves for the wild-type and mutant proteins with and without metal ions were measured by monitoring the changes in CD at 220 nm as a function of temperature in the range of 30–90 °C. The CD unfolding curves for both the wild-type and mutant proteins showed an apparent two-state denaturation (Fig. 3B). As displayed in Table 2, the melting temperature (Tm) of the wild-type is 7 °C higher than the mutant proteins without the metal ions indicating that the disulphide bond plays an important role in the stability of the apo-protein. This also suggests that the mutant proteins start to unfold at lower temperature than the wild-type. The Tm of the wild-type holo-protein is 2 °C higher than that of the apo (Table 2) suggesting that the metal ions marginally contribute in the stability of the wild-type protein. But in both the mutant proteins, the Tm of the holo is 4 °C higher than that of the apo indicating that in the absence of the disulphide bond the metal ions have relatively larger role in the stability of the mutant proteins compared to the wild-type. But it is obvious that the presence of both the disulphide bond and the metal ions are important for the overall stability of the protein. The increase in the stability with both the disulphide bond and the metal ions may be due to the tertiary structure of the protein. In conclusion, the loss of activity in Co2+-reconstituted wildtype H. pylori arginase upon addition of a reducing agent DTT is not due to the involvement of a disulphide bond in catalysis, as the mutants lacking the disulphide bond are found to be catalytically active. The loss of activity may be explained due to the interaction of DTT with Co2+ ions so that free metal ions are not available to form holo-protein. This is supported by the Mn2+reconstituted protein, which exhibited a marginal loss of catalytic activity in both the wild-type and mutant proteins. This explains why mammalian and other bacterial arginases are not sensitive to the reducing agent DTT with Mn2+ as a metal cofactor. Furthermore, we show that the disulphide bond is important for the overall stability and folding of the protein. The presence of the metal ions and the disulphide bond together contribute in the overall stability of the protein than alone. Acknowledgments The work was supported by the National Institute of Immunology and Defence Research and Development Organisation, India. We thank Dr. Sanjeev Galande, National Center of Cell Science,

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