Engineered Citrobacter freundii methionine γ-lyase effectively produces antimicrobial thiosulfinates

Morozova, Elena A.; Kulikova, Vitalia V.; Rodionov, A. N.; Revtovich, Svetlana V.; Anufrieva, Natalya V.; Demidkina, Tatyana V.

doi:10.1016/j.biochi.2016.07.007

Cited by 26 publications

(32 citation statements)

References 30 publications

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“…The replacement allowed us to obtain an effective catalyst for the production of thiosulfinates . It was shown that the catalytic efficiency of wild ‐ type Clostridium sporogenes MGL in the β‐elimination reaction of (±)‐S‐ethyl‐ l ‐cysteine sulfoxide and (±)‐alliin is almost two to six times higher than that of C. freundii MGL . In the present work, we prepared the mutant form of C. sporogenes MGL with the replacement of cysteine 115 by histidine, determined its kinetic parameters in the γ‐ and β‐elimination reactions with characteristic substrates and S‐substituted l ‐cysteine sulfoxides, and evaluated the in vitro antibacterial activity of the sulfoxide/MGL couple against Gram‐positive and Gram‐negative bacteria and clinical isolates from mice.…”

Section: Introductionmentioning

confidence: 92%

“…To avoid the observed 31% inactivation of the enzyme in the β‐elimination reaction of alliin , we prepared the mutant form of C. freundii MGL with the replacement of cysteine 115 by histidine. The replacement allowed us to obtain an effective catalyst for the production of thiosulfinates . It was shown that the catalytic efficiency of wild ‐ type Clostridium sporogenes MGL in the β‐elimination reaction of (±)‐S‐ethyl‐ l ‐cysteine sulfoxide and (±)‐alliin is almost two to six times higher than that of C. freundii MGL .…”

Section: Introductionmentioning

confidence: 99%

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Mutant formC115HofClostridium sporogenesmethionine γ‐lyase efficiently cleavesS‐Alk(en)yl‐l‐cysteine sulfoxides to antibacterial thiosulfinates

et al. 2016

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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.

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Section: Introductionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Mutant formC115HofClostridium sporogenesmethionine γ‐lyase efficiently cleavesS‐Alk(en)yl‐l‐cysteine sulfoxides to antibacterial thiosulfinates

et al. 2016

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“…Isolation of the enzyme, determination of its activity, synthesis of S-substituted L- cysteine sulfoxides, and production of thiosulfinates were carried out as previously described [6]. The concentrations of thiosulfinates were determined according to [8].…”

Section: Methodsmentioning

confidence: 99%

“…We have shown that thiosulfinates can be obtained using methionine γ-lyase (MGL, [EC 4.4.1.11]) ( Scheme ). Thiosulfinates formed by the cleavage of S-allyl- L -cysteine, S-methyl- L -cysteine, and S-ethyl- L -cysteine sulfoxides catalyzed by both wild-type MGL and its more efficient mutant form, C115H, inhibit the growth of Gram-positive and Gram-negative bacteria [6], including P. aeruginosa isolated from murine intestine [7]. …”

Section: Introductionmentioning

confidence: 99%

Antibacterial Effect of Thiosulfinates on Multiresistant Strains of Bacteria Isolated from Patients with Cystic Fibrosis

Kulikova¹,

Chernukha²,

Morozova³

et al. 2018

Acta Naturae

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The multiresistance of A. ruhlandii 155B, B. cenocepacia 122, and P. aeruginosa 48B strains isolated from patients with cystic fibrosis was established. The antibacterial effect of allicin, dimethyl thiosulfinate, and dipropyl thiosulfinate on multidrug-resistant strains was shown. Thiosulfinates can have both bacteriostatic and bactericidal effects depending on the microorganism and the concentration. The studied thiosulfinates may be candidates for the development of alternative antibiotic drugs to treat infections caused by multidrug-resistant pathogens.

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“…E. coli BL21(DE3) cells containing the plasmid with the gene of MGL from C. novyi were grown and the enzyme was purified as described in ref. . The concentration of homogeneous protein was determined by the absorbance at 278 nm, the extinction coefficient (

A_{278}^{0.1 %}

) being 0.8 .…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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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.

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Engineered Citrobacter freundii methionine γ-lyase effectively produces antimicrobial thiosulfinates

Cited by 26 publications

References 30 publications

Mutant formC115HofClostridium sporogenesmethionine γ‐lyase efficiently cleavesS‐Alk(en)yl‐l‐cysteine sulfoxides to antibacterial thiosulfinates

Mutant formC115HofClostridium sporogenesmethionine γ‐lyase efficiently cleavesS‐Alk(en)yl‐l‐cysteine sulfoxides to antibacterial thiosulfinates

Antibacterial Effect of Thiosulfinates on Multiresistant Strains of Bacteria Isolated from Patients with Cystic Fibrosis

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

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