Front Matter

et al., 1995),Ricinus communisL. (Wang et al., 1994; Xu et al., 1997), andCory-

nebacterium ulcerans(McNamara et al., 1995). ForArabidopsis, four types of PLD

were identified (Pappan et al., 1997a,b; 1998; Qin et al., 1997, 1999). Two of these

(PLDband PLDc) are activated by phosphatidylinositol 4,5-bisphosphate similarly

to several mammalian PLD isoforms. Two forms of PLD were cloned and expressed

also from man (Hammond et al., 1995; Lopez et al., 1998), mouse (Colley et al.,

1997a,b) and rat (Park et al., 1997; Kodaki and Yamashita, 1997). From rice and

maize, cDNAs coding for PLD were isolated and analyzed (Ueki et al., 1995). In

cabbage, two isoforms of PLD were identified on the basis of their cDNAs (Pan-

nenberg et al., 1998) with 85 % (PLD1) and 83 % (PLD2) amino acid sequence

identity to PLDaofArabidopsis thaliana. Both isoforms contain eight Cys resi-

dues. Recently, Kim et al. (1999) have described the cloning and expression of a

PLD from cabbage, which corresponds to the sequence of PLD2. InSaccharomyces

cerevisiaean open-reading frame on the chromosome XI was identified encoding a

protein with PLD activity (Waksman et al., 1996). Corresponding deletion mutants

showed that in fact this sequence is responsible for the formation of PLD in yeast

(Ella et al., 1996; Waksman et al., 1996) and resulted in finding a second structurally

unrelated type of PLD in this organism (Waksman et al., 1997). This form might

correspond to the PLD described by Mayr et al. (1996). A PLD gene was also cloned

fromCandida albicans(Kanoh et al., 1998), showing highest homology to PLD from

Saccharomyces cerevisiae.

On the basis of the numerous available DNA sequences coding for PLD, several

structural features and evolutionary ancestors of PLDs could be derived (Ponting and

Kerr, 1996; Sung et al., 1997; reviewed in Morris et al., 1996; Wang, 1997; Wakelam

et al., 1997; Waite, 1999). Although homology between plant, yeast and human

genes of PLD is low, four homologous regions termed as regions I, II, III, and

IV can be defined. PLDs fromStreptomyceshave homologies with short parts of

the regions I, II, and IV, whereas region III is absent in the enzyme from these spe-

cies. Sequence homologies have been found also with cardiolipin synthases and

phosphatidylserine synthases, which both catalyze a reaction including also the

transfer of a phosphatidyl residue. Several more proteins can be assigned to this

family of enzymes, also termed as PLD superfamily, e.g., several endonucleases,

poxvirus envelope proteins or the murine toxin fromYersinia pestis. The hallmark

of all these proteins is the HXKX 4 DX 6 G(G/S) motif containing conserved His, Lys,

Asp, Gly and/or Ser residues. This motif, however, is not absolutely conserved but

allows small variations. All enzymes with this so-called HKD motif catalyze the

cleavage of phosphodiester bonds. Therefore, this motif is suggested to participate

in the active site. For endonuclease fromSalmonella typhimurium(Gottlin et al.,

1998), human PLD (Sung et al., 1997) and the murine toxin fromYersinia pestis

(Rudolph et al., 1999), studies by site-directed mutagenesis proved His and Lys

residues of the HKD motif as being essential for activity. As yet, all PLDs analyzed

contain the HKD motif in duplicate, which are shown for selected enzymes from

plants and microorganisms in Figure 6.

For PLD from rat brain, deletion mutants provided direct evidence that both HKD

motifs are necessary for enzyme activity (Xie et al., 1998). Recently, the crystal

structure of a recombinant form of the corresponding endonuclease fromSalmonel-

la typhimuriumhas been determined (Stuckey and Dixon, 1999). This is the first

230 12 Phospholipases Used in Lipid Transformations