C-terminal loop of Streptomyces phospholipase D has multiple functional roles

Abstract

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.

Figure 1.

Primary structures of chimeras G, I, chimera G mutants, and parental PLD, TH-2PLD, and PLDP, and their transphosphatidylation and hydrolytic activities. (A) The schematic primary structures of PLDs (upper) and amino acid sequence of the regions related to the catalytic reaction (lower) are shown. The regions derived from TH-2PLD and PLDP are indicated in gray and black, respectively. The numbers correspond to TH-2PLD in the amino acid sequence. (B) SDS-PAGE of purified chimera G mutants. Lane M indicates low-molecular-weight marker proteins (molecular weights, 94,000, 67,000, 43,000, 30,000, 20,100 and 14,400). Each sample was applied at 1.5 μg per lane on a 15% acrylamide gel. The arrow indicates the position of purified PLDs. (C) Specific activities of PLDs in transphosphatidylation (upper) and hydrolytic (lower). Transphosphatidylation activity was measured at pH 5.5 with 13 mM PpNP. Hydrolytic activity was determined at pH 5.5 with 2 mM PpNP. Data are expressed as mean ± SD of three independent experiments.

Figure 2.

Identification of amino acid residues related to transphosphatidylation and hydrolytic activities. (A) Primary structures of chimera G, chimera G mutants, and chimera I. (B) Specific activities of chimera G, chimera G mutants, and chimera I in transphosphatidylation (upper) and hydrolytic (lower). Transphosphatidylation activity was measured at pH 5.5 with 13 mM PpNP. Hydrolytic activity was determined at pH 5.5 with 2 mM PpNP. Data are expressed as mean ± SD of three independent experiments.

Figure 3.

Hydrolytic activities of PLDs toward phosphatidylcholine in monomers, micelle, and vesicles. The specific activities of chimera G and mutant G-FH were measured with 5 mM diC₄PC, diC₇PC, or 10 mM POPC in acetate buffer (pH 5.5). Data are expressed as mean ± SD of three independent experiments. The activities of mutant G-FH were significantly different from those of chimera G toward all substrate forms (*P < 0.01, **P < 0.001, ***P < 0.0001, Student's t-test).

Figure 4.

Transphosphatidylation activity of PLDs toward different chain-length phosphatidylcholines. The reaction was performed for 10 min using 10 mM diC₇PC or POPC in acetate buffer (pH 5.5) containing 4 mM CaCl₂, and then the resultant products were analyzed by TLC. Relative PLD activity was determined by measuring the intensity of the spot corresponding to phosphatidylethanol using Scion Image software, and indicated as chimera G activity toward diC₇PC or POPC. The relative activities of mutant G-FH were significantly different from those of chimera G toward diC₇PC and POPC (*P < 0.01, **P < 0.001, Student's t-test).

Figure 5.

Effects of temperature on hydrolytic activities of PLDs. (A) The optimum temperature for PLD hydrolytic activity was determined for 10 min at pH 5.5. Relative PLD activity was calculated with respect to the maximum PLD activity. (B) The thermostability of PLDs was measured for 10 min at pH 5.5. Residual PLD activity was calculated as a ratio to a sample at 15°C. Data are expressed as mean ± SD of three independent experiments.

Figure 6.

Tolerance of PLD against organic solvents. The time course of PLD activity was measured using 50% benzene (A) or ethyl acetate (B). Residual enzyme activity was calculated as a ratio to a sample without an organic solvent. The residual activity of mutant G-FH was significantly different from that of chimera G with ethyl acetate (*P < 0.0001, Student's t-test).

Figure 7.

Selectivities of chimera G (A) and mutant G-FH (B) in transphosphatidylation from POPC to POPG. The reaction was performed using 10 mM POPC with 0.5 M glycerol in acetate buffer (pH 5.5) containing 4 mM CaCl₂ for 10 min, 1 h, 3 h, and 6 h, followed by TLC. Relative contents were determined by measuring the intensities of the spots corresponding to phosphatidic acid, phosphatidylglycerol, and phosphatidylcholine using Scion Image software.

Figure 8.

(A) Overall structure of TH-2PLD using Swiss-PDB viewer based on crystal structure of PMFPLD. The two regions related to enzyme activities are indicated in green (residues 188–203 of TH-2PLD) and orange (residues 425–442 of TH-2PLD). The identified key residues are indicated in red. The N- and C-terminal HKD motifs are shown in light blue and purple, respectively. (B) The local environment around the identified key residues (188, 191, 426, and 438 of TH-2PLD) is represented. The identified key residues are indicated in red. The light-green circle indicates the predicted pocket for the recognition of phospholipids.