Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis

Abstract

A kinetic comparison of the hydrolase and transferase activities of two bacterial phospholipase D (PLD) enzymes with little sequence homology provides insights into mechanistic differences and also the more general role of Ca(2+) in modulating PLD reactions. Although the two PLDs exhibit similar substrate specificity (phosphatidylcholine preferred), sensitivity to substrate aggregation or Ca(2+), and pH optima are quite distinct. Streptomyces sp. PMF PLD, a member of the PLD superfamily, generates both hydrolase and transferase products in parallel, consistent with a mechanism that proceeds through a covalent phosphatidylhistidyl intermediate where the rate-limiting step is formation of the covalent intermediate. For Streptomyces chromofuscus PLD, the two reactions exhibit different pH profiles, a result consistent with a mechanism likely to involve direct attack of water or an alcohol on the phosphorus. Ca(2+), not required for monomer or micelle hydrolysis, can activate both PLDs for hydrolysis of PC unilamellar vesicles. In the case of Streptomyces sp. PMF PLD, Ca(2+) relieves product inhibition by interactions with the phosphatidic acid (PA). A similar rate enhancement could occur with other HxKx(4)D-motif PLDs as well. For S. chromofuscus PLD, Ca(2+) is absolutely critical for binding of the enzyme to PC vesicles and for PA activation. That the Ca(2+)-PA activation involves a discreet site on the protein is suggested by the observation that the identity of the C-terminal residue in S. chromofuscus PLD can modulate the extent of product activation.

Figure 1.

500 MHz ¹H spectra (2.9–3.8 ppm) of 5 mM PI SUVs (in 20 mM imidazole, pH 7.0, with 1 mM Ca²⁺, 23°C) as a function of incubation time (in minutes) with 135 μg S. chromofuscus rPLD. Note the appearance of the sharp myo-inositol peaks (identified by carbon number) in the spectrum.

Figure 2.

Specific activity (μmole min⁻¹ mg⁻¹) of (A) Streptomyces sp. PMF PLD (open circles) and S. chromofuscus rPLD (filled circles) toward diC₇PC in 50 mM Tris HCl, pH 8.0, as a function of substrate concentration. Error bars indicate standard deviations in activity for each PC concentration. (B) Specific activity (μmole min⁻¹ mg⁻¹) of Streptomyces sp. PMF PLD cleavage of diC₆PC as a function of substrate concentration. The arrows indicate the CMC of the pure PC.

Figure 3.

(A) Rates of diC₄PA (filled circles), diC₄PMe (filled squares), and diC₄PG (open circles) production (μmole min⁻¹ mg⁻¹) by Streptomyces sp. PMF PLD (4 μg) as a function of CH₃OH concentration at fixed (5 mM) diC₄PC (assay mixtures also contained 5 mM glycerol from the enzyme stock). (B) Rates of diC₄PA (filled circles), diC₄PMe, and diC₄PG production (open circles) from diC₄PC (5 mM) as function of pH with 5 mM glycerol and 50 mM CH₃OH; (+) represents the mole fraction transferase products (diC₄PG and diC₄PMe) generated by Streptomyces sp. PMF PLD as a function of pH.

Figure 4.

(A) Rate of diC₄PA (filled circles) and diC₄PMe (open circles) production by S. chromofuscus rPLD from 5 mM diC₄PC in 50 mM Tris HCl, 5 mM Ca²⁺, pH 7.5, as a function of added CH₃OH. Enzyme specific activity for generating both products is shown as (X). (B) The pH dependence of the rate of diC₄PA production in the absence (+) and the presence (filled circles) of 2 M CH₃OH; the rate of diC₄PMe (open circles) production in the presence of 2 M CH₃OH is also shown. (C) Mole fraction of PMe produced as a function of pH with 5 mM diC₄PC, 5 mM Ca²⁺ and 2 M methanol, pH 8.0.

Figure 5.

Effect of Ca²⁺ concentration on the (A) hydrolysis and (B) transphosphatidylation reactions of S. chromofuscus rPLD. Assay conditions included 5 mM diC₄PC, 6 M CH₃OH, and 9 μg rPLD. The empty rectangles represent assays in the absence of Ca²⁺; the filled rectangles represent assays with 5 mM Ca²⁺; diagonally striped rectangles represent assays with 20 mM Ca²⁺ added.