Abstact

Phospholipase D (PLD) catalyzes the hydrolysis of phospholipids to generate phosphatidic acid and free head groups such as choline. Among bacterial PLD enzymes, Streptomyces chromofuscus PLD (SchPLD), a member of the alkaline phosphatase D (PhoD) superfamily, exhibits unique Ca(2+)-dependent phospholipase activity. Here, we determined the crystal structure of a PhoD-type PLD from S. avermitilis (SaPLD) at a 2.2-A resolution, which shares 86% sequence identity with SchPLD. The structure revealed the conserved Fe-Ca-Ca catalytic center characteristic of PhoD enzymes. In addition, we identified novel Ca(2+) binding sites surrounding the active site pocket. SaPLD exhibited negligible activity in the absence of Ca(2+) but showed strong activation in the presence of Ca(2+), consistent with previous observations for SchPLD. The overall structure of SaPLD lacks the C-terminal alpha-helix that covers the active site in Bacillus subtilis PhoD, resulting in an expanded hydrophobic cleft suited for bulky phospholipid substrates binding. Molecular dynamics modeling with phosphatidylcholine (PC) indicated that its two oleoyl chains fit well within this cleft, and that the choline head group is accommodated by a distinct cavity formed by Asn217, Leu346, and Asn357. This cavity geometry likely disfavors phosphatidylethanolamine or phosphatidylserine, explaining the preference for PC substrates. These findings provide the first structural insights into the Ca(2+)-stabilized expanded active site of a PhoD-type PLD and clarify the molecular basis for its phospholipid specificity.