First enzymological characterization of selenocysteine β-lyase from a lactic acid bacterium, Leuconostoc mesenteroides.

Abstract

We succeeded in expressing selenocysteine β-lyase (SCL) from a lactic acid bacterium, Leuconostoc mesenteroides LK-151 (Lm-SCL), in the soluble fractions of Escherichia coli Rosetta (DE3) using a novel expression vector of pET21malb constructed by ourselves that has both maltose binding protein (MBP)- and 6 × His-tag. Lm-SCL acted on L-selenocysteine, L-cysteine, and L-cysteine sulfinic acid but showed a high preference for L-selenocysteine. The kcat and kcat/Km values of Lm-SCL were determined to be 108 (min-1) and 42.0 (min-1・mM-1), respectively, and this was enough catalytic efficiency to suggest that Lm-SCL might also be involved in supplying elemental selenium from L-selenocysteine to selenoproteins like other SCLs. The optimum temperature and optimum pH of Lm-SCL were determined to be 37°C and pH 6.5, respectively. Lm-SCL was stable at 37-45°C and pH 6.5-7.5. Lm-SCL was completely inhibited by the addition of hydroxylamine, semicarbazide, and iodoacetic acid. The enzyme activity of Lm-SCL was decreased in the presence of various metal ions, especially Cu2+. The quaternary structure of Lm-SCL is a homodimer with a subunit molecular mass of 47.5kDa. The similarity of the primary structure of Lm-SCL to other SCLs from Citrobacter freundii, Escherichia coli, humans, or mouse was calculated to be 47.0, 48.0, 12.5, or 24.0%, respectively. Unlike Ec-SCL, our mutational and molecular docking simulation studies revealed that C362 of Lm-SCL might also catalyze the deselenation of L-selenocysteine in addition to the desulfuration of L-cysteine.