A novel acid phosphatase excreted by Penicillium funiculosum that hydrolyzes both phosphodiesters and phosphomonoesters with aryl leaving groups

An acid phosphatase has been purified from the culture broth of Penicillium funiculosum by procedures including SP-Sephadex column chromatography and Sephacryl S-200 gel filtration. The phosphatase appears to be a 76 kDa heterodimer composed of 51 and 26 kDa subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes both phosphodiesters and phosphomonoesters, but only those with aryl leaving groups. At the early phase of degradation of bis-p-nitrophenyl phosphate by the enzyme, inorganic phosphate and the intermediary product, p-nitrophenyl phosphate, are liberated at approximately the same rate. This indicates that the intermediary phosphomonoester produced on the enzyme is further hydrolyzed in situ or dissociates into the medium at approximately the same probability.     

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

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 k_cat and k_cat/K_m 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 47.5 kDa. 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.