IVAN V. KUSHKEVYCH*

Institute of Animal Biology of NAAS of Ukraine, Lviv, Ukraine

* Corresponding author: I.V. Kushevych, Institute of Animal Biology of NAAS of Ukraine, Lviv, Ukrain; e-mail: ivan.kushkevych@gmail.com

Submitted 18 June 2014, revised 27 March 2015, accepted 13 April 2015

Abstract

Intestinal sulfate-reducing bacteria reduce sulfate ions to hydrogen sulfide causing inflammatory bowel diseases of humans and animals. The bacteria consume lactate as electron donor which is oxidized to acetate via pyruvate in process of the dissimilatory sulfate reduction. Pyruvate-ferredoxin oxidoreductase activity and the kinetic properties of the enzyme from intestinal sulfate-reducing bacteria Desulfovibrio piger and Desulfomicrobium sp. have never been well-characterized and have not been yet studied. In this paper we present for the first time the specific activity of pyruvate-ferredoxin oxidoreductase and the kinetic properties of the enzyme in cell-free extracts of both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains. Microbiological, biochemical, biophysical and statistical methods were used in this work. The optimal temperature (+35°C) and pH 8.5 for enzyme reaction were determined. The spectral analysis of the puri­fied pyruvate-ferredoxin oxidoreductase from the cell-free extracts was demonstrated. Analysis of the kinetic properties of the studied enzyme was carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period) and maximum velocity of the pyruvate-ferredoxin oxidoreductase reaction (Vmax) were defined. Michaelis constants (Km) of the enzyme reaction were calculated for both intestinal bacterial strains. The studies of the kinetic enzyme properties in the intestinal sulfate-reducing bacteria strains in detail can be prospects for clarifying the etiological role of these bacteria in the develop­ment of inflammatory bowel diseases.

Key words: kinetic analysis, inflammatory bowel diseases, pyruvate ferredoxin oxidoreductase, sulfate-reducing bacteria

Introduction

Intestinal sulfate-reducing bacteria are often isolated from the gut of healthy humans and persons with ulcer­ative colitis and inflammatory bowel diseases (Gibson et al., 1991; Barton and Hamilton, 2010). A greater number of these bacteria is found mainly in sick people (Cummings et al., 2003; Gibson et al., 1991). In the pres­ence of sulfate, lactate in human intestine contributes to the intensive bacteria growth and the accumulation of their final metabolism product, hydrogen sulfide, which is toxic, mutagenic and cancerogenic to epithelial intes­tinal cells (Pitcher and Cummings, 2003; Gibson et al., 1991; Kushkevych, 2012a). The increased number of sulfate-reducing bacteria and the intensity of dissimila­tory sulfate reduction in the gut can cause inflamma­tory bowel diseases of humans and animals (Cummings et al., 2003; Gibson et al., 1991; Kushkevych, 2012b).
Lactate is the most common substrate used by the species belonging to the sulfate-reducing bacteria (Kushkevych, 2012a). This compound is oxidized to acetate via pyruvate. The type of enzyme present in these microorganisms appears to be a pyruvate-ferre­doxin oxidoreductase, as can be deduced from the low potential electron carriers, ferredoxin and flavodoxin, which serve as electron acceptors for the enzyme (Akagi, 1967; Hatchikian et al., 1979; Guerlesquin et al.,1980). In strict anaerobes microorganisms, pyruvate is oxidatively decarboxylated by pyruvate oxidoreduc­tase (EC 1.2.7.1). Pyruvate ferredoxin oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 (Akagi, 1967; Barton and Hamil­ton, 2010; Kushkevych, 2012a).

The reaction of this enzyme has been most exten­sively studied in the forward (oxidative decarboxyla­tion) direction beginning with a series of seminal stud­ies published in 1971 by Raeburn and Rabinowitz which have isolated and characterized pyruvate-ferredoxin oxidoreductase. They have also demonstrated thatlow potential electron donors, like reduced ferredoxin,can drive the reductive carboxylation of acetyl-CoA (Raeburn and Rabinowitz, 1971).
As far as we are aware, pyruvate-ferredoxin oxidoreductase from intestinal sulfate-reducing bacteria D. piger and Desulfomicrobium sp. has never been well-characterized. In the literature there are a lot of data on pyruvate-ferredoxin oxidoreductase in various organ­isms as well as in sulfate-reducing bacteria isolated from environment (Akagi, 1967; Barton and Hamilton, 2010; Hatchikian et al., 1979; Furdui et al., 2000; Garczarek et al., 2007; Guerlesquin et al., 1980; Zeikus et al., 1977; Raeburn and Rabinowitz, 1971; Uyeda and Rabinowitz, 1971; Ma et al., 1997; Meinecke, et al., 1989; Pieulle et al., 1995). However, data on the activity of this enzyme from intestinal sulfate-reducing bacteria D. piger and Desul­fomicrobium sp. have not yet been reported.

The aim of this work was to study pyruvate-ferre­doxin oxidoreductase activity in cell-free extracts of intestinal sulfate-reducing bacteria D. piger Vib-7 and Desulfomicrobium sp. Rod-9 and to carry out the kinetic analysis of enzymatic reaction.
The aim was accomplished using microbiological, biochemical, biophysical methods, and statistical pro­cessing of the results; the obtained data were compared with those from the literature.

Experimental
Materials and Methods

The objects of the study were sulfate-reducing bacte­ria D. piger Vib-7 and Desulfomicrobium sp. Rod-9 iso­lated from the human large intestine and identified by sequence analysis of the 16S rRNA gene (Kushkevych, 2013; Kushkevych et al., 2014).

Bacterial growth and cultivation. Bacteria were grown in a nutrition-modified Kravtsov-Sorokin’s liquid medium (Kushkevych, 2013). Before seeding bacteria in the medium, 0.05 ml/l of sterile solution of Na2S × 9H2O (1%) was added. A sterile 10 N solution of NaOH (0.9 ml/l) in the medium was used to provide the final pH 7.2. The medium was heated in boiling water for 30 min in order to obtain an oxygen-free medium, and then cooled to +30°C. The bacteria were grown for 72 hours at +37°C under anaerobic conditions. The tubes were brim-filled with medium and closed to pro­vide anaerobic conditions.

Obtaining cell-free extracts. Cells were harvested at the beginning of the stationary phase, suspended in 10 mM Tris-HCl buffer in a 1/1 ratio (w/v) at pH 7.6, and disrupted using a Manton-Gaulin press at 9000 psi. The extract was centrifuged at 15,000 g for 1 h; the pel­let was then used as sedimentary fraction, and the supernatant obtained was termed the soluble fraction (Gavel et al., 1998). The soluble extract constituted by the supernatant was used as the source of the enzyme. This extract was subjected to further centrifugation at 180,000 g for 1 h to eliminate the membrane fraction. A pure supernatant, containing the soluble fraction, was then used as cell-free extract. Protein concentration in the cell-free extracts was determined by the Lowry method (Lowry et al., 1951).

Assays for pyruvate-ferredoxin oxidoreductase activity. The pyruvate-ferredoxin oxidoreductase was assayed and purified as described in paper (Pieulle et al., 1995). The enzyme activity was routinely deter­mined spectrophotometrically by following the reduc­tion of methyl viologen as previously described (Zeikus et al., 1977). All enzyme assays were performed under anaerobic conditions at +35°C using serum-stoppered cuvettes. Samples of enzyme were made anaerobic by flushing the solution with argon as previously reported (Fernandez et al., 1985). The reaction mixture contain­ing 50 μmol Tris-HCl (pH 8.5), 10 μmol sodium pyru­vate, 0.1 μmol sodium coenzyme A, 2 μmol methyl viologen and 16 μmol dithioerythritol, in a final vol­ume of 1.0 ml, was bubbled with argon for 20 min and the cell was then incubated at +30°C. The reaction was started by injection of pyruvate-ferredoxin oxidoreduc­tase into the assay cuvette using a gastight syringe and the absorbance at 604 nm was followed. Rates of methyl viologen reduction were calculated using an absorption coefficient of 13.6 mM–1 × cm–1. A regenerating system was used to determine the Km for coenzyme A as pre­viously described (Meinecke et al., 1989). One unit of enzyme activity was defined as the amount of enzyme, which catalyzes the oxidation of 1 μmol of pyruvate or the reduction of 2 μmol of methyl viologen per min under the specified conditions. Specific enzyme activity was expressed as U × mg–1 protein. Michaelis constant (Km) for pyruvate-ferredoxin oxidoreductase reaction has been determined by substrate (pyruvate and coen­zyme A). In order to maintain the concentration of oxi­dized ferredoxin, a recycling system consisting of spin­ach ferredoxin-NADP reductase (5 μg/assay) (Sigma) and NADP+ (5 mM) was used. The overall rate was measured by the appearance of NADPH. The activity of the studied enzyme in the cell-free extracts of both bacterial strains at different temperature (from +20°C to +45°C) and pH (in the range from 5.0 to 10.0) in the incubation medium was measured. Spectral analysis of the purified enzyme was carried out as previously described (Pieulle et al., 1995).

Kinetic analysis. Kinetic analysis of the enzyme reaction was performed in a standard incubation medium (as it was described above) with modified physical and chemical characteristics of the respective parameters (incubation time, substrate concentration, temperature and pH). The kinetic parameters character­izing the pyruvate-ferredoxin oxidoreductase reaction are the initial (instantaneous) reaction velocity (V0), maximum velocity of the reaction (Vmax), maximum amount of the reaction product (Pmax) and characteristic reaction time (time half saturation) were determined. The amount of the reaction product was calculated stoi­chiometrically. The kinetic parameters characterizing pyruvate-ferredoxin oxidoreductase reactions such as Michaelis constant (Km) and maximum reaction velocity of substrate decomposition were determined by Lineweaver-Burk plot (Keleti, 1988). For analysis of the substrate kinetic mechanism of pyruvate-ferredoxin oxidoreductase, initial velocities were measured under standard assay conditions with different substrate con­centrations. The resulting data were also analyzed by global curve fitting in SigmaPlot (Systat Software, Inc.) to model the kinetic data for rapid equilibrium rate equations describing ordered sequential, V=(Vmax [A] [B])/(KA KB+KB [A]+[A] [B]), and random sequential, V=(Vmax [A] [B])/(α KA KB+KB [A]+KA [B]+[A] [B]), kinetic mechanisms, where V is the initial velocity, Vmax is the maximum velocity, KA and KB are the Km values for substrates A and B, respectively, and α is the interac­tion factor if the binding of one substrate changes the dissociation constant for the other (Segal, 1975).

Statistical analysis. Kinetic and statistical calcula­tions of the results were carried out using the software MS Office and Origin computer programs. The research results were treated by the methods of variation statis­tics using Student t-test. The equation of the straight line that the best approximates the experimental data was calculated by the method of least squares. The abso­lute value of the correlation coefficient r was from 0.90 to 0.98. The significance of the calculated parameters of line was tested by Fisher’s F-test. The accurate approxi­mation was when P ≤ 0.05 (Bailey, 1995).

Results and Discussion

Specific activity of pyruvate-ferredoxin oxidoreduc­tase, an important enzyme in the process of organic compounds oxidation in sulfate-reducing bacteria, was measured in different fractions obtained from D. piger Vib-7 and Desulfomicrobium sp. Rod-9 cells (Table I).

Table I
Pyruvate-ferredoxin oxidoreductase activity in different fractions obtained from the bacterial cells

Sulfate-reducing bacteria	Specific activity of the enzyme (U×mg-1 protein)
	Cell-free extract	Individual fractions
		Soluble	Sedimentary
Desulfovibrio piger Vib-7	1.24 ± 0.127	1.11 ± 0.114	0
Desulfomicrobium sp. Rod-9	0.48 ± 0.051**	0.37 ± 0.033***	0

Comment: The assays were carried out at a protein concentration of 43.57 μg/ml (for D. piger Vib-7) and 41.94 μg/ml (for Desulfomicrobium sp. Rod-9). Enzyme activity was deter­mined after 20 min incubation. Statistical significance of the values M ± m, n = 5; **P < 0.01, ***P < 0.001, compared to D. piger Vib-7 strain

Results of our study showed that the highest spe­cific activity of the enzyme was detected in cell-free extracts (1.24 ± 0.127 and 0.48 ± 0.051 U × mg–1 protein for D. piger Vib-7 and Desulfomicrobium sp. Rod-9,respectively). The slightly lower values of activity of pyruvate-ferredoxin oxidoreductase were determined in the soluble fraction compared to cell-free extracts. Its values designated 1.11 ± 0.114 U × mg–1 protein for D. piger Vib-7 and 0.37 ± 0.033 U × mg–1 protein for Desulfomicrobium sp. Rod-9. The enzyme activity in sedimentary fraction was not observed.
The effect of temperature and pH of the reaction mixture on pyruvate-ferredoxin oxidoreductase activity in the cell-free extracts of the sulfate-reducing bacteria was studied (Fig. 1). The maximum specific activity for both bacterial strains was determined at +35°C. The highest enzyme activity of pyruvate-ferredoxin oxidoreductase for D. piger Vib-7 and Desulfomicrobium sp. Rod-9 was measured at pH 8.5.

 

Thus, temperature and pH optimum of this enzyme was +35°C and pH 8.5, respectively. An increase or decrease in temperature and pH led to a decrease of the activity of studied enzyme in the cell-free bacterial extracts of the sulfate-reducing bacteria. The enzyme activity exhibited typical bell-shaped curves as a func­tion of temperature and pH.
Next task of this study was to carry out a spectral analysis of the purified pyruvate-ferredoxin oxidore­ductase from the cell-free extracts of D. piger Vib-7 and Desulfomicrobium sp. Rod-9. The absorption maxima were 317 and 423, 316 and 425 nm for pyruvate-ferre­doxin oxidoreductase from D. piger Vib-7 and Desulfomicrobium sp. Rod-9, respectively (Fig. 2).

 

Ten-minute incubation of the enzyme with 0.75 mM sodium pyru­vate led to a slight decrease in absorption maxima. The same peaks of absorption as without addition of sodium pyruvate were observed. However, the signifi­cant decrease in absorption spectra after the addition of 0.75 mM sodium pyruvate and 0.1 mM coenzyme A in the incubation medium was registered. The absorp­tion peaks was no observed (Fig. 2A). The spectroscopic analyses of oxidized and reduced pyruvate-ferredoxin oxidoreductase from D. piger Vib-7 and Desulfomicro­bium sp. Rod-9 strains were also carried out (Fig. 2B).

Similar data on the absorption spectra of pyruvate-ferredoxin oxidoreductase from Desulfovibrio africanus were obtained by Pieulle et al. (1995). The authors described the ultraviolet-visible spectrum of studied enzyme which was typical of an iron-sulfur protein with a broad absorbance band around 400 nm and a shoul­der in the 315 nm region (Pieulle et al., 1995). Iron and acid-labile sulfide content, as well as the absorp­tion coefficient at 400 nm suggest the presence of six[4Fe-4S] clusters per molecule of enzyme. The absorp­tion band at 400 nm was partially bleached after addi­tion of dithionite; this indicates only partial reduction of the protein, if one considers that full reduction of iron-sulfur clusters should lead to about 50% decrease of the absorption band. Pyruvate reduced the enzyme slightly, whereas pyruvate and CoASH produced a more pronounced reduction of the protein than that obtained with dithionite (Pieulle et al., 1995).

To study the characteristics and mechanism of pyruvate-ferredoxin oxidoreductase reaction, the ini­tial (instantaneous) reaction velocity (V0), maximum velocity of the reaction (Vmax), maximum amount of reaction product (Pmax) and reaction time (τ) were defined. Dynamics of reaction product accumulation was studied for investigation of the kinetic parameters of pyruvate-ferredoxin oxidoreductase (Fig. 3). Experimental data showed that the kinetic curves of pyruvate-ferredoxin oxidoreductase activity have a saturation tendency (Fig. 3A).

 

Analysis of the results allows to reach the conclusion that the kinetics of pyruvate-ferredoxin oxidoreductase activity in the sulfate-reducing bacteria was consistent to the zero-order reaction in the range of 0–10 min (the graph of the dependence of product formation on the incuba­tion time was almost linear in this interval of time). Therefore the duration of the incubation of bacterial cells extracts was 10 min in subsequent experiments.
The amount of the product of pyruvate-ferredoxin oxidoreductase reaction in the D. piger Vib-7 was the higher (36.28 ± 3.59 μmol × mg–1 protein) compared to the Desulfomicrobium sp. Rod-9 (14.95 ± 1.48 μmol × mg–1 protein) in the entire range of time factor. The basic kinetic properties of the reaction in the sulfate-reducing bacteria were calculated by lineari­zation of the data in the {P/t; P} coordinates (Fig. 3B, Table II).

Table II
Kinetic parameters of the pyruvate-ferredoxin oxidoreductase from intestinal sulfate-reducing bacteria

Kinetic parameters	Sulfate-reducing bacteria
	Desulfovibrio piger Vib-7	Desulfomicrobium sp. Rod-9
V0 (µmol×min-1×mg-1 protein)	4.15 ± 0.43	1.37 ± 0.12***
Pmax (µmol×mg-1 protein)	36.28 ± 3.59	14.95 ± 1.48**
(min)	8.74 ± 0.88	10.89 ± 1.11

Comment: V0 is initial (instantaneous) reaction velocity; Pmax is maxi­mum amount (plateau) of the product of reaction; τ is the reaction time (half saturation period). Statistical significance of the values M ± m, n = 5; **P < 0.01, ***P < 0.001, compared to the D. piger Vib-7 strain.

The kinetic parameters of pyruvate-ferredoxin oxi­doreductase from both D. piger Vib-7 and Desulfomi­crobium sp. Rod-9 were significantly different. Values of initial (instantaneous) reaction velocity (V0) for the enzyme was calculated by the maximal amount of the product reaction (Pmax). As shown in Table II, V0 for pyruvate-ferredoxin oxidoreductase reaction was slightly higher (4.15 ± 0.43 μmol × min–1 × mg–1 pro­tein) in D. piger Vib-7 compared to Desulfomicrobium sp. Rod-9 (1.37 ± 0.12 μmol × min–1 × mg–1 protein). In this case, the values of the reaction time (τ) were more similar for the studied enzyme in both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 strains. Based on these data, it may be assumed that the D. piger Vib-7 can consume lactate ion much faster in their cells than a Desulfomicrobium sp. Rod-9. Moreover, this hypo­thetical assumption can be also confirmed by obtained data on maximal velocities of accumulation of the final reaction products, where Vmax for enzyme reaction in D. piger Vib-7 were also more intensively compared to Desulfomicrobium sp. Rod-9 (Table III).

Table III
Kinetic parameters of pyruvate-ferredoxin oxidoreductase reaction

Kinetic parameters	Sulfate-reducing bacteria
	Desulfovibrio piger Vib-7	Desulfomicrobium sp. Rod-9
VmaxPyruvate (µmol×min-1×mg-1 protein)	2.54 ± 0.261	0.89 ± 0.092***
KmPyruvate (mM)	2.72 ± 0.283	2.55 ± 0.245
VmaxCoA (µmol×min-1×mg-1 protein)	2.51 ± 0.248	0.81 ± 0.076***
KmCoA (μM)	0.54 ± 0.052	0.42 ± 0.044

Comment: Vmax is maximum velocity of the enzyme reaction; Km is Michaelis constant which was determined by substrate (pyruvate and coenzyme A). Statistical significance of the values M ± m, n = 5; ***P < 0.001, compared to the D. piger Vib-7 strain.

The kinetic analysis of pyruvate-ferredoxin oxidoreductase reaction depending on concentration of sub­strate (pyruvate and coenzyme A) was carried out. The increasing pyruvate concentrations from 0.5 to 5.0 mM and coenzyme A concentrations from 0.1 to 1.0 μM caused a monotonic rise of the studied enzyme activ­ity and the activity was maintained on unchanged level (plateau) under substrate concentrations over 5.0 mM and 1.0 μM, respectively. (Fig. 3C, E). Curves of the dependence {1/V; 1/[S]} were distinguished by the tan­gent slope and intersect the vertical axis in one point (Fig. 3D, F). The basic kinetic parameters of pyruvate-ferredoxin oxidoreductase activity in D. piger Vib-7 and Desulfomicrobium sp. Rod-9 were identified by lineariza-tion of the data in the Lineweaver-Burk plot (Table III).
Calculation of the kinetic parameters of enzyme activity indicates that the maximum velocities (Vmax) of pyruvate and coenzyme A in the D. piger Vib-7 and Desulfomicrobium sp. Rod-9 were significantly differ­ent from each other. However, it was observed a cor­relative relationship between VmaxPyruvate and VmaxCoA in both intestinal bacterial strains. Michaelis constants (Km) of pyruvate-ferredoxin oxidoreductase reaction were identified for pyruvate and coenzyme A. The val­ues of Km were quite similar for pyruvate (2.72 ± 0.283, 2.55 ± 0.245 mM) and coenzyme A (0.54 ± 0.052, 0.42 ± 0.044 μM) in both D. piger Vib-7 and Desulfomi­crobium sp. Rod-9 strains, respectively.

The obtained parameters of pyruvate-ferredoxin oxidoreductase reaction in D. piger Vib-7 are consist­ent with previously described data by Pieulle et al. for the activity of pyruvate-ferredoxin oxidoreductase from D. africanus. The apparent Km for pyruvate and coenzyme A were also 2.5 mM and 0.5 μM, respectively and the Vmax values were 10240 min–1 and 5890 min–1, respectively. The apparent Km for methyl viologen was found to be 0.5 mM in the presence of 10 mM and 0.1 mM of pyruvate and CoASH, respectively. Kinetics studies done with the enzyme and a slight decrease in the affinity for pyruvate and in the catalytic activity (Km of 5.5 mM and Vmax of 4810 min–1) were reported (Pieulle et al., 1995).

Furdui and Ragsdale (2000) have described the pyruvate-ferredoxin oxidoreductase from the Clostri-dium thermoaceticum. The Michaelis-Menten param­eters for pyruvate synthesis by the enzyme were:Vmax 1.6 unit/mg, KmAcetyl-CoA 9 μM. The intracellular con­centrations of acetyl-CoA, CoASH, and pyruvate were also measured (Furdui and Ragsdale, 2000).
Pyruvate-ferredoxin oxidoreductatse, an important enzyme in process of dissimilatory sulfate reduction and organic compounds oxidation in sulfate-reducing bacte­ria, carries out the central step in oxidative decarboxyla­tion of pyruvate to acetyl-CoA (Kushkevych, 2012a): Garczarek et al. (2007) have purified this enzyme from Desulfovibrio vulgaris Hildenborough as part of a systematic characterization of as many multiprotein complexes as possible for this organism (Garczarek et al., 2007).

Thus, based on the obtained studies results and according to the kinetic parameters of pyruvate-ferredoxin oxidoreductatse reaction for both bacterial strains, we have concluded that the enzyme activity, V0 and Vmax were significantly higher in the D. piger Vib-7 cells than Desulfomicrobium sp. Rod-9. How­ever, Michaelis constants were quite similar for pyru­vate (2.72 ± 0.283, 2.55 ± 0.245 mM) and coenzyme A (0.54 ± 0.052, 0.42 ± 0.044 μM) in both bacterial strains. The maximum enzyme activity for both strains was determined at +35°C and at pH 8.5. These data cor­respond to conditions which are present in the human large intestine from where the bacterial strains were isolated. Perhaps such conditions favor intensive devel­opment of the D. piger and Desulfomicrobium sp. bacte­rial strains in the gut. The kinetic parameters of enzyme reaction are depended on the substrate concentration. The studies of the pyruvate-ferredoxin oxidoreductatse in the process of dissimilatory sulfate reduction and kinetic properties of this enzyme in the D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal strains,their production of acetate in detail can be a perspective for clarification of their etiological role in the develop­ment of the humans and animals bowel diseases. These studies might help in predicting the development of diseases of the gastrointestinal tract, by providing fur­ther details on the etiology of bowel diseases, which are very important for the clinical diagnosis of these disease types.

Acknowledgements
The author expresses his gratitude to Dr. Roman Fafula, M. Sc., Ph.D. from Biophysics Department of Faculty of Pharmacy, Danylo Halytsky Lviv National Medical University (Ukraine) for his assis­tance in performing the kinetic analysis and critical reading of the manuscript.

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