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Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine reSeArcH ArtIcLeS Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii E. A. Morozova1, V. V. Kulikova1, D. V. Yashin2, N. V. Anufrieva1, N. Y. Anisimova3, S. V. Revtovich1, M. I. Kotlov4, Y. F. Belyi4, V. S. Pokrovsky3, T. V. Demidkina1* 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova Str., 32, Moscow, Russia, 119991 2Institute of Gene Biology, Russian Academy of Sciences, Vavilova St., 34/5, Moscow, Russia, 119334 3Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe sh., 24, Moscow, Russia, 115478 4Gamaleya Research Institute of Epidemiology and Microbiology, Gamaleya Str., 18, Moscow, Russia, 123098 *E-mail: [email protected], [email protected] Received 04.03.2013 Copyright © 2013 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT The steady-state kinetic parameters of pyridoxal 5’-phosphate-dependent recombinant methionine γ-lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingiva- lis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in com- parison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4–1.3 U/ml), PC-3 (IC50=0.1–0.4 U/ml), and MCF7 (IC50=0.04–3.2 U/ml) turned out to be the most sensitive cell lines. KEYWORDS kinetic parameters; methionine γ-lyase; pathogenic microorganisms; oligomeric structure; cytotox- icity. ABBREVIATIONS DTT – dithiothreitol; His-tag – poly-histidine fragment; MGL – methionine γ-lyase; MSC – mes- enchymal stromal cells; PLP – pyridoxal 5’-phosphate. INTRODUCTION sponding mercaptans, pyruvic acid, and ammonia [1]. Methionine γ-lyase (MGL) [EC 4.4.1.11] is a pyridoxal the enzyme was isolated from Pseudomonas putida, 5’-phosphate-dependent enzyme that catalyzes the Aeromonas sp., Clostridium sporogenes, Porphyrom- γ-elimination reaction of L-methionine, yielding me- onas gingivalis, Brevibacterium linens BL2, Citrobacter thyl mercaptan, α-ketobutyric acid, and ammonia: freundii, and several others, from eukaryotic protozoa Trichomonas vaginalis and Entamoeba histolytica, and from fungi Aspergillus flavipes [2]. this enzyme is ab- sent in mammalian cells but is present in pathogenic bacteria, such as Aeromonas sp. [3], C. sporogenes [4], In addition to the physiological reaction, the enzyme P. gingivalis [5], and in pathogenic protozoa E. histo- catalyzes the β-elimination reaction of L-cysteine lytica [6], T. vaginalis [7], which allows one to consider and its S-substituted derivatives, yielding the corre- the enzyme as a potential target for novel antibiotics. 92 | ActA nAturAe | VOL. 5 № 3 (18) 2013 reSeArcH ArtIcLeS the enzyme is of interest as an anticancer agent, since the activity of the preparations in the γ-elimination the growth of malignant cells of various origins (unlike reaction was determined by the rate of formation of the growth of normal cells) is accompanied by obliga- α-ketobutyric acid in the coupled reaction with D-2- tory methionine utilization [8]. the possibility of de- hydroxy isocaproate dehydrogenase under the condi- velopment of an anticancer agent based on MGL from tions described in [17]. One unit of enzyme activity was P. putida has been demonstrated in vitro and in vivo defined as the quantity of MGL catalyzing the forma- [9–12]. Antitumor activity of the enzyme from A. fla- tion of 1.0 µM/min α-ketobutyrate at 30°c. the specific vipes with respect to several human tumors was ob- activities of the enzyme from C. tetani, C. sporogenes, served in vivo [13]. Several kinetic parameters of the P. gingivalis and C. freundii were 16.6, 12.8, 5.0, and recombinant MGL derived from the pathogenic micro- 10.2 u/mg, respectively. organisms C. tetani (causative agent of tetanus), C. spo- cleavage of the His-tag fragment was performed in rogenes (causative agent of gas gangrene and enteritis), a reaction with thrombin. the reaction mixture (1 ml) and P. gingivalis (causative agent of periodontitis) were containing 10 mg of the enzyme in a 50 mM potassium determined in the present study; the data on the cy- phosphate buffer, pH 8.0, 1 mM DTT, 0.05 mM PLP, totoxic activity of the enzyme from these sources and and 100 units of thrombin were incubated for 24 h at from C. freundii were analyzed. 4°c. the product was then purified by gel filtration on a Superdex 200 column (Ge Healthcare, Sweden) equili- EXPERIMENTAL brated with a 50-mM potassium phosphate buffer, pH 8.0, containing 1 mM Dtt and 0.05 mM PLP. the ho- Bacterial cultivation and purification of the enzyme mogeneity of the preparation was determined by elec- Escherichia coli BL21 (De3) cells containing the MGL trophoresis under denaturing conditions. genes from C. sporogenes, C. tetani, and P. gingivalis in the plasmid pet-28a [14] were cultured in an “induc- Determination of the oligomeric composition of ing” medium [15] at 37°c under stirring (180 rpm) for MGL from C. tetani, C. sporogenes, and P. gingivalis 24 h. the cells were harvested by centrifugation and the molecular weights of the enzymes from C. teta- stored at –80°c. cell disruption and the removal of nu- ni, C. sporogenes, and P. gingivalis were determined cleic acids were performed according to [16]. After the for the enzymes containing His-tag, and after the separation of nucleic acids, the preparations were trans- thrombin-facilitated cleavage of the tag, followed by ferred to a 50 mM potassium phosphate buffer, pH 8.0, gel filtration on a Superdex 200 10/300 GL column (Ge containing 0.05 mM pyridoxal 5’-phosphate (PLP) using Healthcare, Sweden). A 50 mM potassium phosphate a “centricon-30 ultrafiltration unit” (Amicon, uSA). buffer, pH 8.0, containing 0.05 mM PLP and 1 mM DTT the polypeptide chains of the enzymes isolated from was used for elution. three sources contained the poly-histidine fragment MGSSHHHHHHSSGLVPrGSH at their n-termini. Determination of the steady-state kinetic the preparations were purified using affinity chroma- parameters of the γ- and β-elimination reactions tography on a column with a ni2+IMAc –Sepharose the steady-state parameters of the γ-and β-elimination sorbent (Ge Healthcare, Sweden); a 10–500 mM imida- reactions were determined by the formation rate of zole gradient in a 50 mM potassium phosphate buffer, α-ketobutyric and pyruvic acids in coupled reactions pH 8.0, containing 0.05 mM PLP was used for MGL elu- with D-2-hydroxy isocaproate dehydrogenase and tion. Fractions with the characteristic spectra of py- lactate dehydrogenase under the conditions described ridoxal 5’-phosphate-dependent enzymes with λmax at in [17] by varying the substrate concentrations in the 420 nm were obtained at an imidazole concentration of reaction mixtures. the data were processed according 25–155 mM. cultivation of the biomass of E. coli BL21 to the Michaelis–Menten equation using the enzfitter (De3) cells containing a plasmid with the MGL gene software program. the molecular weights of the en- from C. freundii and purification of the enzyme was zyme subunits with allowance for His-tag (which were carried out as per [17]. the concentrations of the pu- equal to 44.04, 44.36, an 44.08 kDa for MGL from C. teta- rified preparations were determined using the coef- ni, C. sporogenes, P. gingivalis, respectively) were used 278 ficient А1% = 0.8 [17]. the purity of the preparations in the calculations. was evaluated using electrophoresis under denatur- ing conditions according to the Laemmli method [18]. Evaluation of in vitro cytotoxicity the bands in the electrophoregrams were identified the cytotoxic activity of MGL of various origins was by coomassie r-250 staining [19] and Western blot- evaluated for the Fisher L5178y lymphadenosis cell ting using the poly-histidine fragment HisProbe-HrP line (a collection of tumor strains from the n.n. Blokhin reagent (thermo scientific, rockford, IL, uSA) [20]. cancer research center), Pc-3 and Lncap human VOL. 5 № 3 (18) 2013 | ActA nAturAe | 93 reSeArcH ArtIcLeS Table 1. Kinetic parameters of MGL from various sources* MGL from P. gingivalis MGL from C. tetani MGL from C. sporogenes MGL from C. freundii** Substrate / kcat, Кm, kcat Km, kcat, Km, kcat /Km, kcat, Km, kcat /Km, kcat, Km, kcat /Km,, s-1 mM M-1s-1 s-1 mM M-1s-1 s-1 mM M-1s-1 s-1 mM M-1s-1 L-Met 3.9 1.77 2.2×103 12 0.947 1.27×104 9.86 0.432 2.28×104 6.2 0.7 8.85×103 S-et-L-Hcy 3.84 0.93 4.13×103 5.89 0.545 1.08×104 7.05 0.278 2.54×104 6.78 0.54 1.26×104 L-Met(SO) 5.05 12.22 4.13×102 2.7 7.07 3.82×102 6.7 33.51 2.0×102 2.52 6.21 4.06×102 S-et-L-cys 8.05 2.17 3.71×103 7.08 0.72 9.83×103 6.3 0.358 1.76×104 5.03 0.17 2.96×104 S-Bzl-L-cys 5.8 1.47 3.94×103 8.5 0.766 1.11×104 10 0.348 2.87×104 8.16 0.18 4.53×104 * The mean squared error of the experiment in the determination of the kinetic parameters did not exceed 10%.
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