Front Matter

(Abousalham et al., 1995), and castor bean (Wang et al., 1993a). Purified microbial

PLDs allowing the determination of molecular data were obtained from several

StreptomycesandStreptoverticilliumstrains (Table 1). Recently, the crystallization

of a PLD fromStreptomyces antibioticushas been achieved successfully (Suzuki et

al., 1999). Highly purified mammalian PLDs were obtained from porcine lung

(Okamura and Yamashita, 1994), murine (Jenco et al., 1998) and human tissues

(Hammond et al., 1997).

Most types of PLD require Ca2+ions for activity, the optimum concentrations

being extremely high (20–100 mM) in case of plant enzymes (Heller, 1978).

The pH optimum is strongly dependent on the Ca2+concentration. For PLD from

soybean, the pH changes from 7.5 at 10 mM CaCl 2 to pH 5.5 at 40 mM CaCl 2

(Abousalham et al., 1995). PLDs are capable of hydrolyzing and transesterifying

a broad range of phospholipids including PC, phosphatidylethanolamine (PE), phos-

phatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositides (PI), lyso-

PC, cardiolipin, and plasmalogens (Heller, 1978), with preferences depending on

the enzyme source and isoform. With respect to thesn-2 position of phospha-

tides, catalysis is stereospecific. PLD from cabbage catalyzes the cleavage of the

naturally occurring PC form much faster than that of its enantiomeric form. This

stereospecificity, however, is completely lost if the 2-acyl group in the substrate

is replaced by an alkyl group (Bugaut et al., 1985). Alkylphosphate esters such

as the antitumor agent hexadecylphosphocholine are also substrates for PLD (Dit-

trich et al., 1996; 1998). The degree of unsaturation influences the substrate speci-

ficity of PLD (Abousalham, 1997). In addition to water, a wide range of alcohols can

serve as phosphatidyl acceptors (see Section 1.5.4). Anionic amphiphiles such as

sodium dodecylsulfate (SDS), triphosphoinositide, monocetylphosphate (Dawson

and Hamington, 1967), phosphatidic acid (Jung et al., 1989), 1,3-diacylglycero-

2-sulfate or 1,3-diacylglycero-2-phosphate (Dittrich et al., 1998) were found to

act as activators of PLD from cabbage, while neutral lipids such as Triton X-100

(Okawa and Yamaguchi, 1975), cholesterol and 1,2-diacylglycerols were reported

to activate PLD fromStreptomyces chromofuscus(Yamamoto et al., 1993; 1995).

Phosphatidic acid or other charged lipids with a free phosphate group are also acti-

vators of this enzyme (Geng et al., 1998). The activation effect was attributed to a

Ca2+-mediated interaction of the compound with PLD at an allosteric site located in

the C-terminal region of the enzyme (Geng et al., 1999). Choline, ethanolamine and

protamine sulfate (Dawson and Hamington, 1967), 1,3-diacylglycero-2-phospho-

cholines (Dittrich et al., 1998; Haftendorn et al., 2000), lysophosphatidylethanol-

amine (Ryu et al., 1997), alkylphosphate esters (Dittrich et al., 1996; 1998) or alu-

minum fluoride (Li and Fleming, 1999) proved to be inhibitors of PLD. Irreversible

inhibition was obtained by 4-chloromercuribenzoate (Yang et al., 1967), diethylpyro-

carbonate (Lee et al., 1989; Secundo et al., 1996) or 4-bromophenacylbromide (Lee

et al., 1989). Reviews on plant, microbial and mammalian PLDs can be found in

Waite (1987).

A remarkable advance in the knowledge on PLDs has started by the intense re-

search into the molecular genetics of PLD. PLD genes have been cloned and ex-

pressed fromStreptomyces chromofuscus(Yoshioka et al., 1990), Streptomyces

acidomyceticus(Hasegawa et al., 1992), Streptomyces antibioticus (Iwasaki et

al., 1994),Corynebacterium pseudotuberculosis(Hodgson et al., 1992; McNamara

12.3 Molecular structure and mechanism of phospholipases used as biocatalysts 229