Recombinant expression and characterization of secretory phospholipaseA₂ isoforms in the tobacco.

Abstract

PhospholipaseA₂(PLA₂) hydrolyzes glycerophospholipids specifically at the sn-2 position to yield free fatty acids and lysophospholipids. In animals, PLA₂s are classified according to the primary structure, Ca²⁺ dependence and cellular localization. Secretory PLA₂ (sPLA₂) is Ca²⁺ dependent and low molecular mass enzyme. Animal sPLA₂ commonly contains Ca²⁺ binding domain and catalytic domain. We have recently cloned two cDNAs encoding sPLA₂ from the tobacco. They also have the Ca²⁺ binding domain and the catalytic domain in their primary structures. In this study, we constructed bacterial expression system for these sPLA₂ and investigated their enzymatic properties. Interestingly, the one hydrolyzed the fatty acyl ester bond at the sn-1 and -2 positions of glycerophospholipids. They essentially required Ca²⁺ for their enzymatic activities. They showed different pH optimum and substrate specificity. We introduced site-directed mutations into some conserved amino acid residues to generate recombinant sPLA₂ mutants and studied their enzymatic properties.