(The) roles of propeptide and accessory protein (PlaB) of phospolipase $A_1$ from serratia sp. MK1

Abstract

A phospholipase $A_1$ operon of Serratia sp.MK1 consists of plaA and plaB coding phospholipase $A_1$ and accessory protein respectively. Serratia.sp. MK1 secretes phospholipase $A_1$ into culture medium where there was two different sizes of the secreted phospholipase $A_1$. N-terminal signal sequence prediction and N-terminal amino acid sequencing of phospholipase $A_1$ showed that phospholipase $A_1$ is synthesized as a preproprotein which had a prepeptide as a signal sequence and a propeptide. The propeptide is supposed to be cleaved off by non-serine extracellular protease after secretion because serine protease inhibitor, PMSF,did not lower the propeptide processing. The size of mature phospholipase $A_1$ was 250 a.a while preproprotein of phospholipase $A_1$ had 320 a.a. We studied roles of propeptide and an accessory protein, PlaB on the restoring of phospholipase $A_1$ activity in the cell. The proprotein of phospholipase $A_1$ showed higher activity than mature phospholipase $A_1$ when expressed intracellularly in E.coli. plaB increased the extracellular phospholipase $A_1$ production when expressed in E.coli with phospholipase $A_1$ gene in two different plasmids. In addition, a refolding efficiency of denatured phospholipase $A_1$ was increased by the addition of PlaB. Accordingly,PlaB seemed to be a molecular chaperone which mediated protein folding and increased phospholipase $A_1$ activity. However, phospholipase $A_1$ activity was lowered in vitro when PlaB was added to the mature phospholipase $A_1$. Finally, we concluded that propeptide and PlaB were intramolecular chaperone which mediated the folding of phospholipase $A_1$ and additionally PlaB acted as an inhibitor. PlaB acted as a chaperone when phospholipase $A_1$ had a propeptide and an inhibitor when phospholipase $A_1$ was a mature protein.