Gene cloning, characterization, and cytotoxic activity of methionine γ‐lyase from Clostridium novyi

Abstract

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 and k /K 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 IC of 1.3 U mL , 4.4 U mL , 1.2 U mL , and 3.4 U mL , 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.

Effects in Cancer Cells of the Recombinant l-Methionine Gamma-Lyase fromBrevibacterium aurantiacum.Encapsulation in Human Erythrocytes for Sustained l-Methionine Elimination

“…The requirement for cofactor is probably owing to weak binding of the apoenzyme to PLP, a feature that may be exhibited by most MGLs (Sun et al, 2003;Yang et al, 2004). However, differences in binding affinities of apoenzyme for cofactor between these enzymes have been reported, e.g., dissociation constant (K D ) values for binding to PLP were found to be 0.62 mM for the MGL from Citrobacter freundii and 14.5 mM for the MGL from Clostridium novyi (Kulikova et al, 2017).…”

Methionine γ-Lyase-Daidzein in Combination with S-Propyl-L-cysteine Sulfoxide as a Targeted Prodrug Enzyme System for Malignant Solid Tumor Xenografts

“…The recombinant C115H MGL was obtained from Escherichia coli BL21 (DE3) cells containing pET28a plasmid with the inserted gene of the mutant form of C. novyi C115H MGL. The cells were grown and the enzyme was isolated as described in [ 29 ]. The assays were performed by measuring the rate of the β-elimination reaction in 100 mM potassium buffer solution, pH 8.0, containing 1 mM EDTA, 0.1 mM PLP, 1 mM DTT and 100 mM S-methyl-L-cysteine [ 29 ].…”

“…The cells were grown and the enzyme was isolated as described in [ 29 ]. The assays were performed by measuring the rate of the β-elimination reaction in 100 mM potassium buffer solution, pH 8.0, containing 1 mM EDTA, 0.1 mM PLP, 1 mM DTT and 100 mM S-methyl-L-cysteine [ 29 ]. The rate of pyruvate formation was determined in the coupled reaction with LDH by reducing the absorption of NADH at 340 nm (ε = 6220 M −1 cm −1 ) at 37 °C.…”

Serine 339 in the Catalysis of γ- and β-Elimination Reactions

“…Метионин-γ-лиаза (МГЛ, [КФ 4.4.1.11]) -ПЛФзависимый фермент, катализирующий реакции γ-элиминирования L-метионина, β-элиминирования L-цистеина и их S-замещенных производных, а также реакции γи β-замещения L-метионина, L-цистеина и их аналогов [6,7]. Фермент найден у патогенных простейших [8,9], в ряде бактерий, в том числе патогенных [10][11][12][13][14], грибах [15] и растениях [16].…”

“…Methionine γ-lyase (MGL, EC 4.4.1.11) is a PLPdependent enzyme that catalyzes the γ-elimination reaction of L-methionine, the β-elimination reaction of L-cysteine , and their S-substituted derivatives, as well as the γ- and β-replacement reactions of L-methionine, L-cysteine, and their analogs [ 6 , 7 ]. The enzyme is found in pathogenic protozoa eukaryotes [ 8 , 9 ], a number of bacteria, in particular pathogenic ones [ 10 , 11 , 12 , 13 , 14 ], fungi [ 15 ], and plants [ 16 ].…”