Association of N- and C-terminal domains of phospholipase D is required for catalytic activity

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X)(4)D, denoted HKD, RPLD1 contains two HKD domains, located in the N- and C-terminal regions. Deletion mutant s of rPLD1 that contained only an N- or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HAD domains were cotransfected with a C-ter minal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N- and C-terminal h alves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 signifi cantly enhanced basal PLD activity while inhibiting the response to phorbol ester, Likewise, the coexpression of this truncated N-terminal half with t he C-terminal half resulted in increased PLD activity. In summary, this stu dy provides direct evidence that the enzymatic activity of rPLD1 requires t he presence of the HRD domains in both the N- and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and thi s may be essential to bring the two HKD domains together to form an active catalytic center, These findings provide new insights into the catalytic me chanism of enzymes of the PLD superfamily.