Redesigning methionine γ-lyase for improved stability and catalytic activity in the β-elimination reaction for the synthesis of thiosulfinates.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are involved in many cellular processes and possess unequalled catalytic versatility. Rational design through site-directed mutagenesis is a powerful strategy for creating tailor-made enzymes for a wide range of biocatalytic applications. PLP-dependent methionine γ-lyase (MGL), which degrades sulfur-containing amino acids, is an encouraging enzyme for many therapeutic purposes - from combating bacterial resistant strains and fungi to antitumor activity. A two-component biosystem MGL/S-alk(en)yl-L-cysteine sulfoxide (an uncommon substrate for this enzyme) produces antimicrobial thiosulfinates during the β-elimination reaction. SH-groups of the enzyme are modified by the products of this reaction, which leads to the inactivation of the enzyme. Successful and efficient rational stabilization of MGL from Clostridium novyi can be achieved using site-directed mutagenesis. We have managed to obtain an improved version of the enzyme better than the natural one regarding the β-elimination reaction. Cys118, Cys184 and Cys273 of MGL from Clostridium novyi were substituted by His and two Ala, respectively. The resulting Cys-del variant had 2-3-fold improved k/K value for conventional β-eliminating substrates and up to 10-fold increase in catalytic efficiency with S-substituted-L-cysteine sulfoxides compared to the wild-type MGL. The Cys-del MGL remained active under the thiosulfinates, which are products of β-elimination reaction of S-alk(en)yl-L-cysteine sulfoxides. Moreover, Cys-del variant proved to be more stable during shelf storage. Thus, we have created an effective enzyme component of the biocatalytic system that is capable of generating antimicrobial drugs - thiosulfinates.