Svetlana V. Revtovich

Structure of Citrobacter freundii l-methionine γ-­lyase

l-Methionine γ-lyase (MGL) is a pyridoxal 5′-phosphate (PLP) dependent enzyme that catalyzes γ-elimination of l-methionine. The crystal structure of MGL from Citrobacter freundii has been determined at 1.9 A resolution. The spatial fold of the protein is similar to those of MGLs from Pseudomonas putida and Trichomonas vaginalis. The comparison of these structures revealed that there are differences in PLP-binding residues and positioning of the surrounding flexible loops.

Methionine γ-lyase: Mechanistic deductions from the kinetic pH-effects: The role of the ionic state of a substrate in the enzymatic activity

We have studied and compared the pH-dependencies of the main kinetic parameters for the alpha,gamma-elimination reactions of methionine gamma-lyase (MGL) of Citrobacter intermedius with natural substrate, l-methionine, with its phosphinic analogue, and for alpha,beta-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k(cat)/K(m) for the reaction with l-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the pK(a) of the substrates amino group, equal to 7.55, is not reflected in the pH-profile of k(cat)/K(m). Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k(cat)/K(m) for reactions with l-methionine and S-methyl-l-cysteine are controlled by a single pK(a) equal to 7.1-7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal pK(a)s of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k(cat)/K(m) for the reactions with S-methyl-l-cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average pK(a) values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both alpha,gamma- and alpha,beta-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the alpha,gamma-elimination, and Tyr 58 - in the alpha,beta-elimination process.

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Background: Speculative chemical mechanism of methionine γ-lyase is formulated, kinetic and structural data concerning elementary stages of physiological reaction are mostly lacking. Results: Pre-steady-state kinetic and structural analysis of the enzyme interaction with inhibitors was performed. Conclusion: Results elucidate the mechanism of intermediate interconversion at initial stages of enzymatic reaction. Significance: The data serve for understanding detailed mechanism of pyridoxal 5′-phosphate-dependent γ-elimination reaction. 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 Å 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.

Alliin is a suicide substrate of Citrobacter freundii methionine γ-lyase: structural bases of inactivation of the enzyme

The interaction of Citrobacter freundii methionine 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.

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.

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

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.

Crystal structure of mutant form Cys115His of Citrobacter freundii methionine γ-lyase complexed with l-norleucine

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 1.45Å 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.