Vitalia V. Kulikova

Tryptophan indole-lyase from Proteus vulgaris: Kinetic and spectral properties

An efficient method for purification of recombinant tryptophanase from Proteus vulgaris was developed. Catalytic properties of the enzyme in reactions with L-tryptophan and some other substrates as well as competitive inhibition by various amino acids in the reaction with S-o-nitrophenyl-L-cysteine were studied. Absorption and circular dichroism spectra of holotryptophanase and its complexes with characteristic inhibitors modeling the structure of the principal reaction intermediates were examined. Kinetic and spectral properties of two tryptophanases which markedly differ in their primary structures are compared. It was found that although the spectral properties of the holoenzymes and their complexes with amino acid inhibitors are different, the principal kinetic properties of the enzymes from Proteus vulgaris and Escherichia coli are analogous. This indicates structural similarity of their active sites.

Role of Arginine 226 in the Mechanism of Tryptophan Indole-Lyase from Proteus vulgaris

In the spatial structure of tryptophanase from Proteus vulgaris the guanidinium group of arginine 226 forms a salt bridge with the 3′-oxygen atom of the coenzyme. The replacement of arginine 226 with alanine using site-directed mutagenesis reduced the affinity of the coenzyme for the protein by one order of magnitude compared to the wild-type enzyme. The catalytic activity of the mutant enzyme in the reaction with L-tryptophan was reduced 105-fold compared to the wild-type enzyme. The rates of the reactions with some other substrates decreased 103-104-fold. The mutant enzyme catalyzed exchange of the C-α-proton in complexes with some inhibitors with rates reduced 102-fold compared to the wild-type enzyme. Absorption and circular dichroism spectra of the mutant enzyme and the enzyme–inhibitor complexes demonstrate that the replacement of arginine 226 with alanine does not significantly affect the tautomeric equilibrium of the internal aldimine, but it leads to an alteration of the optimal conformation of the coenzyme–substrate intermediates.

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.