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Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme catalyzing γ-elimination in l-methionine. Pyridoxal 5'-phosphate-dependent enzymes have unique spectral properties that allow to monitor sequential formation and decomposition of various intermediates via the detection of absorbance changes. The kinetic mechanism of the γ-elimination reaction catalyzed by Citrobacter freundii MGL was elucidated here by fast stopped-flow kinetic analysis. Single-wavelength detection of characteristic absorbance changes enabled us to compare transformations of intermediates in the course of the reaction with different substrates. The influence of various γ-substituents in the substrate on the formation of key intermediates was estimated. Kinetic isotope effects of α- and β-protons were determined using deuterium-substituted l-methionine. Contributions of amino acid residues Tyr113 and Tyr58 located in the active site on the formation and decomposition of reaction intermediates were identified too. α-Aminocrotonate formation is the rate-limiting step of the enzymatic γ-elimination reaction. Kinetic isotope effects strongly support concerted reaction mechanisms of transformation between an external aldimine and a ketimine intermediate as well as a ketimine intermediate and an unsaturated ketimine.

Serine 339 of the active site of Citrobacter freundii methionine -lyase (MGL) is a conserved amino acid in most pyridoxal 5-phosphate-dependent enzymes of the cystathionine -lyase subclass, to which MGL belongs. The reaction mechanism of the MGL-catalyzed -elimination reaction is poorly explored. We replaced serine 339 with alanine using site-directed mutagenesis. The replacement of serine 339 with alanine led to a significant (by two orders of magnitude) decrease in efficiency in the catalysis of the - and -elimination reactions by the mutant form of the enzyme. The exchange rates of the C-- and C--protons in the amino acids in complexes consisting of the enzyme and competitive inhibitors decreased by one-two orders of magnitude. The spectral characteristics of the mutant form indicated that the replacement did not lead to significant changes in the conformation and tautomerism of MGL internal aldimine. We crystallized the holoenzyme and determined its spatial structure at 1.7 resolution. The replacement of serine 339 with alanine did not affect the overall course of the polypeptide chain of the MGL subunit and the tetrameric enzyme structure. An analysis of the obtained kinetic and spectral data, as well as the known spatial structures of C. freundii MGL, indicates that serine 339 is necessary for efficient catalysis of - and -elimination reactions at the stage of C--proton abstraction from the external aldimine, the -elimination reaction at the stages of coenzyme C4-atom protonation, and C--proton abstraction from a ketimine intermediate.

Therapeutic enzymes used for the treatment of a wide range of human disorders often suffer from suboptimal pharmacokinetics and stability. Engineering approaches such as encapsulation in micro- and nanocarriers, and replacements of amino acid residues of the native enzyme provide significant potential for improving the performance of enzyme therapy. Here, we develop a nanodelivery system on the base of polyion complex vesicles (PICsomes) that includes methionine γ-lyase (MGL) as a therapeutic enzyme. We have two strategies for using the enzyme: first, methionine γ-lyase is an anticancer agent removing l-methionine from plasma, second, the binary system methionine γ-lyase/S-alk(en)yl-l-cysteine sulfoxides is effective in enzyme prodrug therapy (EPT). Various lengths polymers were synthesized, and two mutant forms of the enzyme were used. The catalytic and pharmacokinetic parameters of the nanoformulations were investigated. The catalytic efficiencies of encapsulated enzymes were comparable to that of native enzymes. Pharmacokinetic analysis has shown that inclusion into PICsomes increases half-life of the enzymes, and they can be safely administered in vivo. The results suggest the further use of encapsulated MGLs for EPT and anticancer therapy, and this strategy could be leveraged to improve the efficiency of enzyme-based therapies for managing serious human diseases.

The methionine dependence is a well known phenomenon in metabolism of cancer cells. Methionine γ-lyase (EC 4.4.1.11, MGL) catalyzes the γ-elimination reaction of L-methionine and thus could effectively inhibit the growth of malignant cells. Recently we have demonstrated that the mutant form of the enzyme C115H MGL can be used as a component of the pharmacological pair enzyme/S-(allyl/alkyl)-L-cysteine sulfoxides to yield thiosulfinates in situ. Thiosulfinates were shown to be toxic to various cancer cell lines. Therefore the application of the enzyme in enzyme pro-drug therapy may be promising. The conjugates of MGL and C115H MGL with polysialic acid were obtained and their kinetic and pharmacokinetic parameters were determined. The formation of polysialic shell around the enzyme was confirmed by atomic force microscopy. The half-life of conjugated enzymes increased 3–6 times compared to the native enzyme. The cytotoxic effect of conjugated MGL against methionine dependent cancer cell lines was increased two times compared to the values for the native enzymes. The anticancer efficiency of thiosulfinates produced by pharmacological pair C115H MGL/S-(allyl/alkyl)-L-cysteine sulfoxides was demonstrated in vitro. The results indicate that the conjugates of MGL with polysialic acid could be new antitumor drugs.

Lung disease caused by Pseudomonas aeruginosa is the leading reason for death in cystic fibrosis patients. Therapeutic efficacy of the pharmacological pairs, the naked/encapsulated mutant form of C...

Interactions of Citrobacter freundii methionine γ-lyase (MGL) with sulfoxides of typical substrates were investigated. It was found that sulfoxides are suicide substrates of the enzyme. The products of the β- and γ-elimination reactions of sulfoxides, thiosulfinates, oxidize three cysteine residues of the enzyme. Three-dimensional structures of MGL inactivated by dimethyl thiosulfinate and diethyl thiosulfinate were determined at 1.46 A and 1.59 A resolution. Analysis of the structures identified SH groups oxidized by thiosulfinates and revealed the structural bases of MGL inactivation. The extent of inactivation of MGL in the catalysis of the β-elimination reaction depends on the length of the «tail» at oxidized Cys115. Oxidation of Cys115 results in MGL incapable to catalyze the stage of methyl mercaptan elimination of the physiological reaction.

Citrobacter freundii methionine γ-lyase (MGL), in addition to the physiological reaction, catalyzes the β-elimination reaction of S-alk(en)yl-L-cysteine sulfoxides to yield thiosulfinates, which have antibacterial activity. We have obtained the mutant form C115H MGL, which cleaves S-alk(en)yl-L-cysteine sulfoxides more effectively than the wild type enzyme does. The binary system MGL/S-alk(en)yl-L-cysteine sulfoxides may be considered as a new pharmacological pair in enzyme prodrug therapy (EPT). Despite of the successful application of this pair in antibacterial studies in vitro, in vivo experiments may lead to several problems typical of therapeutic proteins including a relatively short-lasting biological activity. To circumvent these problems, we have investigated several approaches to improve safety and efficacy of the enzyme component of the pharmacological pair. This included covalent attachment of poly(ethylene glycol) to the enzyme, its encapsulation in liposomes and polymeric vesicles (PICsomes). The steady-state and pharmacokinetic parameters of modified/encapsulated enzyme were determined. It was demonstrated that the encapsulation in PICsomes prolongs in vivo stability of C115H MGL to over 42 h compared to PEGylated enzyme (3 h). Antibacterial activity of binary system (“pharmacological pair”) modified/encapsulated enzyme/S-alk(en)yl-L-cysteine sulfoxides was tested and remained the same as for the naked enzyme. Thus, the usage of MGL-loaded PICsomes as enzymatic nanoreactors in ETP to produce antimicrobial thiosulfinates is promising.

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

The gene NT01CX_1210 of pathogenic bacterium Clostridium novyi annotated as encoding O-acetylhomoserine sulfhydrylase was cloned and expressed in Escherichia coli. The gene product having O-acetylhomoserine sulfhydrylase activity was purified to homogeneity. The protein showed molecular mass of approximately 184 kDa for the native form and 46 kDa for the subunit. The enzyme catalyzes the γ-substitution reaction of O-acetylhomoserine with maximum activity at pH 7.5. Analysis of C. novyi genome allowed us to suggest that there is only one way for the synthesis of l -methionine in the bacterium. The data obtained may provide the basis for further study of the role of OAHS in Clostridium bacteria and an ascertainment of its mechanism.

We presented results of monotherapy and combination therapy of transplantable murine tumor models using methionine-gamma-lyase (MGL) and pyridoxine hydrochloride. We studied MGL from Clostridium sporogenes and Citrobacter freundii. We used Lewis lung carcinoma (LLC), melanoma B16, leukemias P388 and L1210 and Fisher lymphadenosis L5178y. Neither monotherapy with MGL nor combination of MGL and pyridoxine demonstrated antitumor activity against P388 and L5178y. In the murine L1210 leukemia model, MGL C. sporogenes injected intraperitoneally in the dose of 2000 U/kg, 11 times with a 12-hour interval increased the life span of mice (ILS=22 %, р=0.035). In the LLC model, the combination of MGL C. sporogenes at a dose of 400 U/kg, i.p., 4 times with a 48-hour interval and pyridoxine at a dose of 250 mg/kg led to tumor growth inhibition (TGI=55 %, р

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits kcat and kcat /Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL-1 , 4.4 U mL-1 , 1.2 U mL-1 , and 3.4 U mL-1 , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017.

PK studies were carried out after a single i.v. administration of 500 and 1000 U/kg by measuring of MGL activity in plasma samples. L-methionine concentration was measured by mass spectrometry. After single i.v. injection of 500U/kg the circulating T1/2 of enzymes in mice varies from 73 to 123min. The AUC0-tinf values determined for MGL 500U/kg from C. freundii, C. tetani and C. sporogenes are 8.21±0.28, 9.04±0.33 and 13.88±0.39U/(ml×h), respectively. Comparison of PK parameters of three MGL sources in the dose of 500U/kg indicated the MGL C. sporogenes to have better PK parameters: clearance 0.83(95%CI: 0.779-0.871) - was lower than C. tetanii 1.27(95%CI: 1.18-1.36) and C. freundii 1.39(95%CI: 1.30-1.49). Mice plasma methionine decreased to undetectable level 10min after MGL 1000 U/kg injection. After MGL C. sporogenes 500U/kg injection plasma methionine level completely omitted after 10min till 6h, assuming the sustainability of negligible levels of methionine (

S-Alk(en)yl-l-cysteine sulfoxides, initially found in plants of the genus Allium, are converted to antimicrobial thiosulfinates by pyridoxal 5'-phosphate(PLP)-dependent alliinase (EC 4.4.1.4). It was found that methionine γ-lyase (MGL, EC 4.4.1.11) catalyzes the β-elimination reaction of (±)-S-alk(en)yl-l-cysteine sulfoxides to yield thiosulfinates. The efficient catalyst for the production of thiosulfinates, C115H mutant MGL, developed in our previous work, cleaves S-alk(en)yl-l-cysteine sulfoxides more effectively than the wild type enzyme. Thiosulfinates generated by the C115H MGL/sulfoxide system have demonstrated growth inhibition of Gram-positive, Gram-negative bacteria and clinical isolates of pathogenic bacteria from mice. In search of a more effective system for production of antibacterial thiosulfinates we synthesized S-substituted analogues of l-cysteine sulfoxide with a longer side chains - (±)-S-propyl-l-cysteine sulfoxide ((±)-propiin) and (±)-S-n-butyl-l-cysteine sulfoxide ((±)-butiin) and determined catalytic parameters of the β-elimination reaction of two sulfoxides. It was found that C115H MGL cleaves (±)-propiin with the highest rate, as compared to other (±)-S-alk(en)yl-l-cysteine sulfoxides. Studies on interaction of the enzyme with (+)- or (-)-S-alk(en)yl-l-cysteine sulfoxides revealed that C115H MGL can decompose both diastereomers equally. The antibacterial activity of the mixture of the mutant MGL with (±)-propiin is comparable with those of the mixtures with S-allyl-l-cysteine sulfoxide (alliin) and S-methyl-l-cysteine sulfoxide (methiin). The results make MGL/sulfoxide system more advantageous in preparing antibacterial thiosulfinates as compared to alliinase-based system, which preferably cleaves naturally occurring (+)-sulfoxides.

Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016.

The problem of resistance to antibiotics requires the development of new classes of broad-spectrum antimicrobial drugs. The concept of pro-drugs allows researchers to look for new approaches to obtain effective drugs with improved pharmacokinetic and pharmacodynamic properties. Thiosulfinates, formed enzymatically from amino acid sulfoxides upon crushing cells of genus Allium plants, are known as antimicrobial compounds. The instability and high reactivity of thiosulfinates complicate their use as individual antimicrobial compounds. We propose a pharmacologically complementary pair: an amino acid sulfoxide pro-drug and vitamin B6 - dependent methionine -lyase, which metabolizes it in the patients body. The enzyme catalyzes the - and -elimination reactions of sulfoxides, analogues of L-methionine and L-cysteine, which leads to the formation of thiosulfinates. In the present work, we cloned the enzyme gene from Clostridium sporogenes. Ionic and tautomeric forms of the internal aldimine were determined by lognormal deconvolution of the holoenzyme spectrum and the catalytic parameters of the recombinant enzyme in the - and -elimination reactions of amino acids, and some sulfoxides of amino acids were obtained. For the first time, the possibility of usage of the enzyme for effective conversion of sulfoxides was established and the antimicrobial activity of thiosulfinates against Gram-negative and Gram-positive bacteria in situ was shown.

In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate “handle” of pyridoxal 5′-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96 A resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 A resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits k

The mutant form of Citrobacter freundii methionine γ-lyase with the replacement of active site Cys115 for His has been found to be inactive in the γ-elimination reaction of methionine while fully active in the γ-elimination reaction of O-acetyl-l-homoserine and in the β-elimination reaction of S-alk(en)yl-substituted cysteines. In this work, the crystal structure of the mutant enzyme complexed with competitive inhibitor, l-norleucine was determined at 1.45A resolution. At the enzyme active site the inhibitor proved to be bound both noncovalently and covalently, which corresponds to the two intermediates of the γ- and β-elimination reactions, Michaelis complex and the external aldimine. Analysis of the structure allowed us to suggest the possible reason for the inability of the mutant enzyme to catalyze the physiological reaction.

The interaction ofCitrobacter freundiimethionine γ-lyase (MGL) and the mutant form in which Cys115 is replaced by Ala (MGL C115A) with the nonprotein amino acid (2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) was investigated. It was found that MGL catalyzes the β-elimination reaction of alliin to form 2-propenethiosulfinate (allicin), pyruvate and ammonia. The β-elimination reaction of alliin is followed by the inactivation and modification of SH groups of the wild-type and mutant enzymes. Three-dimensional structures of inactivated wild-type MGL (iMGL wild type) and a C115A mutant form (iMGL C115A) were determined at 1.85 and 1.45 Å resolution and allowed the identification of the SH groups that were oxidized by allicin. On this basis, the mechanism of the inactivation of MGL by alliin, a new suicide substrate of MGL, is proposed.

Pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) catalyzes the β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides to thiosulfinates, which possess antimicrobial activity. Partial inactivation of the enzyme in the course of the reaction occurs due to oxidation of active site cysteine 115 conserved in bacterial MGLs. In this work, the C115H mutant form of Clostridium sporogenes MGL was prepared and the steady-state kinetic parameters of the enzyme were determined. The substitution results in an increase in the catalytic efficiency of the mutant form towards S-substituted l-cysteine sulfoxides compared to the wild type enzyme. We used a sulfoxide/enzyme system to generate antibacterial activity in situ. Two-component systems composed of the mutant enzyme and three S-substituted l-cysteine sulfoxides were demonstrated to be effective against Gram-positive and Gram-negative bacteria and three clinical isolates from mice. © 2016 IUBMB Life, 68(10):830-835, 2016.

Antimicrobial activity of thiosulfinates in situ produced by mixtures of Citrobacter freundii methionine γ-lyase (MGL) with new substrates, l-methionine and S-(alkyl/allyl)-l-cysteine sulfoxides has been recently demonstrated (Anufrieva et al., 2015). This opens a way to the rational design of a new biotechnologically relevant antimicrobial drug producer. To increase the efficiency of the enzyme toward sulfoxides, the mutant forms of MGL, with the replacements of active site cysteine 115 with alanine (C115A MGL) and histidine (C115H MGL) were obtained. The replacement of cysteine 115 by histidine results in the loss of activity of the mutant enzyme in the γ-elimination reaction of physiological substrate, whereas the activity in the β-elimination reaction of characteristic substrates persists. However, the catalytic efficiency of C115H MGL in the β-elimination reaction of S-substituted l-cysteine sulfoxides is increased by about an order of magnitude compared to the wild type MGL. The antibacterial activity of C115H MGL mixtures with a number of sulfoxides was assessed against Gram-positive and Gram-negative bacteria. The bacteriostatic effect was more pronounced against Gram-positive than against Gram-negative bacteria, while antibacterial potential proved to be quite similar. Thus, the mutant enzyme C115H MGL is an effective catalyst, in particular, for decomposition of sulfoxides and the pharmacological couples of the mutant form with sulfoxides might be new antimicrobial agents.

Methionine γ-lyase (MGL) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the γ-elimination reaction of L-methionine. The enzyme is a promising target for therapeutic intervention in some anaerobic pathogens and has attracted interest as a potential cancer treatment. The crystal structure of MGL fromClostridium sporogeneshas been determined at 2.37 Å resolution. The fold of the protein is similar to those of homologous enzymes fromCitrobacter freundii,Entamoeba histolytica,Pseudomonas putidaandTrichomonas vaginalis. A comparison of these structures revealed differences in the conformation of two flexible regions of the N- and C-terminal domains involved in the active-site architecture.

The three-dimensional structure of the external aldimine of Citrobacter freundii methionine γ-lyase with competitive inhibitor glycine has been determined at 2.45 A resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of Cα-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of Cα-proton to C4′-atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic Cα-protons of glycine may be explained on the basis of external aldimine structure. The exchange of Cα- pro -(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from Cα-atom of glycine to C4′-atom of the cofactor. The exchange of Cα- pro -(S)-proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of β-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid.

The steady-state kinetic parameters of pyridoxal 5-phosphate-dependent recombinant methionine -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in - and -elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the -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 -lyase from C. sporogenes and C. tetani in comparison 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.