O-Acetylhomoserine Sulfhydrylase from Clostridioides difficile: Role of Tyrosine Residues in the Active Site.

O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5'-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.

Kinetic and pharmacokinetic characteristics of therapeutic methinoninе γ-lyase encapsulated in polyion complex vesicles.

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.