Cloning and Characterization of the Acid Lipase from Castor Beans

• Published in: The Journal of biological chemistry 2004-10, Vol.279 (44), p.45540-45545
• Main Author: Eastmond, Peter J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cloning, Molecular; Escherichia coli; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Molecular Sequence Data; Rhizomucor miehei; Ricinus communis - enzymology; Substrate Specificity; Triglycerides - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M408686200

Castor bean endosperm contains a well known acid lipase activity that is associated with the oil body membrane. In order to identify this enzyme, proteomic analysis was performed on purified oil bodies. A ∼60-kDa protein was identified (RcOBL1), which shares homology with a lipase from the filamentous fungus Rhizomucor miehei . RcOBL1 contains features that are characteristic of an α/β-hydrolase, such as a putative catalytic triad ( S DH) and a conserved pentapeptide (G X S X G) surrounding the nucleophilic serine residue. RcOBL1 was expressed heterologously in Escherichia coli and shown to hydrolyze triolein at an acid pH (optima ∼4.5). RcOBL1 can hydrolyze a range of triacylglycerols but is not active on phospholipids. The activity is sensitive to the serine reagent diethyl p -nitrophenyl phosphate, indicating that RcOBL1 is a serine esterase. Antibodies raised against RcOBL1 were used to show that the protein is restricted to the endosperm where it is associated with the surface of oil bodies. This is the first evidence for the molecular identity of an oil body-associated lipase from plants. Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.