tertiary structure of a PLD family member, and conclusions to common structural
features of these enzymes have been derived. The crystal structure suggests that
residues from two HKD-motifs form a single active site. The conserved His residue
of one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoen-
zyme intermediate, whereas the His residue of the other motif probably functions as a
general acid in the cleavage of the phosphodiester bond. Moreover, the structure of
the endonuclease suggests that the conserved Lys residues are involved in phosphate
binding. The suggested reaction mechanism includes a phosphatidyl – enzyme inter-
mediate, and is in accordance with previous stereochemical analyses of substrates
and products of cabbage PLD proving that catalysis by PLD proceeds with retention
of configuration at the phosphorus (Bruzik and Tsai, 1984). From studies on the
hydrolysis of PC by PLD with H 218 O (Holbroock et al., 1991) it was concluded
that the P–O bond rather than the O–C bond of the choline phosphate moiety is
cleaved. Recent studies by site-directed mutagenesis and recombinant protein frag-
ments of PLD fromStreptomyces antibioticus(Iwasaki et al., 1999) also resulted in
the conclusion that the two HKD motifs in PLD are not functionally equivalent. The
His residue acting as catalytic nucleophile and binding to the phosphatidyl group of
the substrate is located on theC-terminal half of PLD, whereas the N-terminal half
did not contain such a nucleophile. The proposed reaction course is demonstrated in
Figure 7. The first step involves the formation of the phosphatidyl – enzyme inter-
mediate, and requires an in-line nucleophilic attack on the phosphorus atom of the
substrate by H442 and protonation of the leaving alcohol moiety by H168 as a gen-
eral acid, while the second step requires deprotonation of a water molecule by the
deprotonated H168 as a general base and another in-line nucleophilic attack on the
phosphorus atom by the deprotonated water. In difference, studies on chemical pro-
tein modification of PLD fromStreptomycessp. (Secundo et al., 1996) suggest that
Lys but not His residues are essential for activity of the enzyme.
232 12 Phospholipases Used in Lipid Transformations
Figure 7. Reaction course of the hydrolysis of phospholipids by PLD. (Mechanism as suggested by
Iwasaki et al., 1999, for PLD fromStreptomyces antibioticus).